Fluorescent dyes, fluorescent dye kits, and methods of preparing labeled molecules

ABSTRACT

The present invention provides methods, compositions, and kits useful in preparing labeled molecules, which are useful in the detection of binding partners.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/454,484, filed on Mar. 18, 2011, which is incorporated herein byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Fluorescent dyes are widely used in biological research and medicaldiagnostics. Fluorescent dyes are superior to conventional radioactivematerials because fluorescent dyes are less expensive and less toxic,and can typically be detected with sufficient sensitivity. Inparticular, a diversity of fluorophores with a distinguishable colorrange has made it more practical to perform multiplexed assays capableof detecting multiple biological targets at the same time. The abilityto visualize multiple targets in parallel is often required fordelineating the spatial and temporal relationships amongst differentbiological targets in vitro and in vivo. In addition, the generation ofa wide range of fluorescent dyes has opened a new avenue for conductinghigh-throughput and automated assays, thus dramatically reducing theunit cost per assay. Moreover, the low toxicity of fluorescent dyesprovides ease of handling in vitro, and also renders it safer forimaging biological activities in vivo.

Fluorescent dyes for applications as described above generally derivefrom joining a dye and an agent that is capable of binding to a giventarget or binding partner. Current processes for joining dyes to suchbinding agents, antibodies being one example, typically requirepurification of the antibody away from standard buffer components, longreaction times, and purification of the labeled antibody once thereaction is complete. Thus, current processes consume valuable time andresources, and there is a need for improvements in the labeling ofbinding agents.

SUMMARY OF THE INVENTION

In various embodiments, a method for preparing a labeled proteincomprises: (a) providing i) a reactive dye, such as an amine-reactivedye, or a reactive detectable label, such as an amine-reactivedetectable label, and ii) a first amine; (b) combining theamine-reactive dye and the first amine, in a buffer, with a samplesolution comprising a target protein, the target protein comprising asecond amine, to form a combined solution; and (c) allowing the combinedsolution to react, thereby producing the labeled protein. For example,the amine-reactive detectable label reacts more strongly with the secondamine than the first amine. In various embodiments, the combinedsolution reacts for less than 3, 2, 1, 0.5 or fewer hours. In variousembodiments, the reaction is essentially complete in about 30 minutes orless. The reactive detectable label may be a fluorescent dye, biotin,digoxin, a hapten or an epitope. In some embodiments, the reactivedetectable label is not a dye. The reactive group of the reactivedetectable label may be an amine-reactive group. In some embodiments,the amine-reactive detectable dye or amine-reactive detectable label hasa molecular weight of less than about 10000, 5000, 3000, 2000, or 1000Da. For example, the amine-reactive detectable dye or amine-reactivedetectable label has a molecular weight of less than about 5000 Da.

In various embodiments, the target protein is an antibody, for examplean antibody fragment, a recombinant antibody, a non-human antibody, achimeric antibody, a humanized antibody, or a fully human antibody.

The buffer may be selected from the group consisting of a sodiumcarbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Trisbuffer, a MOPS buffer, a HEPES buffer, or a combination thereof. Thebuffer may be alkaline. For example, the pH of the buffer may be in therange from 7 to 10 or the pH of the combined solution may be greaterthan 7.8.

In various embodiments, the first amine is a primary or secondary amine,such as an aliphatic amine capable of competing in the labelingreaction. For example, the first amine may betris(hydroxymethyl)aminomethane (Tris), a weak primary aliphatic amine.The amine, such as Tris, may be present in the buffer at a concentrationof greater than 20 mM, or may be formulated to be present in thecombined solution at a concentration of greater than 1 mM, for exampleformulated to about 10 mM.

The reactive dye may comprises one or more activated esters, such as aN-hydroxy succinimidyl ester, N-hydroxy sulfosuccinimidyl ester,p-sulfo-tetrafluorophenol ester, or a combination thereof. The reactivedye may comprise one or more sulfonate groups and/or one or morewater-soluble polymer groups such as polyethylene glycol. In variousembodiments, the water-soluble polymer group has a molecular weight of300-10000 Daltons, or 450-5000 Daltons. Reactive dyes may be selectedfrom the group consisting of: a CF dye, an Alexa Fluor dye, a DyLightDye, a Cy dye, an IRDye, a HiLyte dye, a sulfonated and/or pegylatedcoumarin dye, a sulfonated and/or pegylated xanthenes dye, a sulfonatedor/pegylated cyanine dye, and a sulfonated and/or pegylated pyrene dye.In various embodiments, the dye has a chemical structure according toany of Compounds 1-128.

In various embodiments, a fixed amount of reactive label is used tolabel a target protein in any amount within a certain range, resultingin a labeled target protein that avoids being either under labeled orover labeled. For example, the sample solution comprises between about 1μg and about 1000 μg, or between about 5 μg and about 200 μg, or betweenabout 5 μg and about 20 μg, or between about 20 μg and about 50 μg, orbetween about 50 μg and about 100 μg, or between about 50 μg and about200 μg, or between about 100 μg and about 200 μg of said target protein.The ratio of amount of protein in microgram (μg) to the amount ofreactive dye in nanomole (nmol) may be from 30:1 to 1:1. The samplesolution may further comprise one or more non-target proteins. Forexample, the ratio of the combined weight of target and non-targetprotein in microgram (μg) to the amount of reactive dye in nanomole(nmol) may be from 30:1 to 1:1. In various embodiments, the ratio of theweight in microgram (μg) of target protein to non-target protein is fromabout 10:1 to about 1:10.

In various embodiments, a method of preparing a protein-proteinconjugate is provided comprises:

(a) providing i) an amine-reactive bifunctional crosslinker additionallycomprising a hydrolysis-resistant functional group; and optionally ii) afirst amine; (b) combining, in a first buffer, the amine-reactivebifunctional crosslinker and, if present, the first amine, with a samplesolution comprising a target protein, the target protein comprising asecond amine, to form a first combined solution; (c) allowing thecombined solution to react, thereby producing a target proteinfunctionalized with a hydrolysis-resistant functional group; (d) in asecond buffer, combining the target protein functionalized with thehydrolysis-resistant functional group with a reporter protein comprisinga hydrolysis-resistant reactive group, wherein said hydrolysis-resistantfunctional group reacts with said hydrolysis-resistant reactive group,thereby forming a protein-protein conjugate. For example, theamine-reactive detectable label reacts more strongly with the secondamine than the first amine; and step (d) is performed withoutpurification of the combined solution from step (c).

In various embodiments, the first buffer is any aqueous buffer having apH from about 7 to about 10. The buffer may comprise one or more primaryor secondary aliphatic amine. For example, the buffer may be abicarbonate buffer comprising Tris.

The amine-reactive group of the bifunctional crosslinker may be anactivated ester, such as succinimidyl ester, and thehydrolysis-resistant functional group of the crosslinker may be selectedfrom an aldehyde, a ketone, an azide, an alkyne, a phosphineparticipating in Staudinger conjugation, a diene, a dienophile, aprotected hydrazino, a protected aminooxy. For example, the crosslinkermay be a bifunctional molecule comprising a succinimidyl ester and analdehyde group.

In various embodiments, as with the target protein labeling using areactive SDL above, the ratio of amount of protein in microgram (μg) tothe amount of the crosslinker in nanomole (nmol) may be from 30:1 to1:1. The sample solution may further comprise one or more non-targetproteins. For example, the ratio of the combined weight of target andnon-target protein in microgram (μg) to the amount of crosslinker innanomole (nmol) may be from 30:1 to 1:1. In various embodiments, theratio of the weight in microgram (μg) of target protein to non-targetprotein is from about 10:1 to about 1:10.

In various embodiments, the reporter protein is generally a proteinmolecule that can produce a detectable signal either by itself or oninteracting with another molecule. For example, protein tags may includebut are not limited to fluorescent proteins, tandem dyes and enzymes. Insome embodiments, the fluorescent proteins are phycobiliproteins, suchas R-phycoerythrine, tandem dyes are fluorescent resonance energytransfer (FRET) dyes typically prepared by covalently labelingfluorescent proteins with one or more synthetic organic dyes, such asthe ones mentioned above. In some embodiments, the enzymes arehorseradish peroxidase or alkaline phosphatase. The hydrolysis-resistantreactive group on the reporter protein is generally a reactive groupcapable of reacting with the hydrolysis-resistant functional group ofthe crosslinker to form a covalent bond. Non-limiting suitablehydrolysis-resistant reactive groups include an aldehyde, a ketone, anazido, an alkyne, a phosphine participating in Staudinger conjugation, adiene, a dienophile, a hydrazone and an oxime.

In various embodiments, the second buffer may be a buffer having a pHfrom about 4 to about 10. According to some embodiments, the secondbuffer may comprise one or more catalyst that facilitates theconjugation of the protein tag to the functionalized target protein. Forexample, the second buffer may be an acidic buffer having a pH fromabout 4 to about 6.8 and comprising aniline or an aniline derivative forcatalyzing an aminooxy to an aldehyde conjugation to form an oximelinkage.

In various embodiments, a pretreatment purification step is excluded.For example, in various embodiments, protein A-based chromatography,size-exclusion chromatography, and ultra membrane filtration are notperformed.

Methods of staining one or more biological targets are also disclosed.Such methods may comprise (a) preparing one or more labeled proteinsaccording to the methods described herein, wherein each of said one ormore labeled proteins comprises a targeting moiety that binds to abinding partner associated with one or more of said biological targets;and, (b) exposing said one or more biological targets to said one ormore labeled proteins, such that said labeled protein binds to saidbinding partner thereby staining said one or more biological targets. Inother embodiments, methods are provided of staining a biological targetcomprising (a) preparing one or more labeled proteins according to themethods described herein, and (b) exposing said one or more biologicaltargets to said one or more labeled proteins, such that said labeledprotein binds to said one or more biological targets thereby stainingsaid one or more biological targets. In various embodiments, step (b) isperformed without purification of said one or more labeled proteins fromstep (a). In various embodiments, the use of a quencher is excluded.Staining may take place in vivo or in vitro, as desired. In variousembodiments, two or more labeled proteins comprise different dyes, suchthat said exposing step (b) renders said different binding partnersoptically distinguishable.

The products of staining may be analyzed by, for example, flowcytometry, western blot, or by microscopy. For example, the products maybe analyzed using a fluorescence activated cell sorter.

In various embodiments, diagnosing a condition of a subject based onanalysis of stained biological targets is performed. Alternatively, amethod for evaluating the efficacy of a test compound is disclosed.

A kit for preparing a labeled protein is provided which may comprise (a)a buffer comprising a first amine, for example a primary or secondaryaliphatic amine; (b) one or more reactive dyes or reactive detectablelabels for labeling one or more target proteins; (c) a storage buffer;and, (d) instructions in one or more than one language. The kitcomprises components according to the methods described herein. Forexample, one or more of dyes may be present as an amine reactive dye.Buffer may be selected from the group consisting of: a sodium carbonatebuffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, aMOPS buffer, a HEPES buffer, or a combination thereof. In variousembodiments, the buffer is alkaline, for example, with a pH in the rangeof 7 to 10. The amine may be tris(hydroxymethyl)aminomethane (Tris),present in a concentration, for example, of greater than 20 mM, or in aconcentration such that upon addition to a protein, the concentrationwould be greater than 20 mM.

Activated esters are encompassed, such as N-hydroxy succinimidyl ester,N-hydroxy sulfosuccinimidyl ester, p-sulfo-tetrafluorophenol ester, or acombination thereof.

A kit for preparing reporter protein-labeled proteins is also providedwhich may comprise (a) an alkaline buffer comprising a first amine, suchas a primary or secondary aliphatic amine; (b) a bifunctionalcrosslinker comprising an amine-reactive group and ahydrolysis-resistant functional group; (c) a reporter proteinfunctionalized with a pairing hydrolysis-resistant reactive group; (d) asecond buffer suitable for the hydrolysis-resistant functional group andhydrolysis-resistant reactive group to react to form a covalent linkage;(e) a storage buffer; and (f) instructions in one or more than onelanguage.

In various embodiments, buffer and dye are present in an amountsufficient to permit labeling of 5 μg-200 μg of a target protein.Storage buffer may be present, for example, with a stabilizer. Suchstabilizers may include bovine serum albumin, gelatin, glycerol, sodiumazide, tris, or a combination thereof.

In various embodiments, the kit includes a separation device forpurifying a labeled target protein. For example, the separation devicemay be a size exclusion chromatography column. In various embodiments, astain stabilizing reagent for enhancing dye fluorescence is present inthe kit.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)),each of which are incorporated by reference for their teachings ofconventional techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows the results of a flow cytometry analysis of cells stainedwith secondary antibodies labeled according to methods of the invention.

FIG. 2 shows the results of a flow cytometry analysis of cells stainedwith secondary antibodies labeled according to methods of the invention.

FIG. 3 shows the results of a flow cytometry analysis of cells stainedwith secondary antibodies labeled according to methods of the invention.

FIG. 4 shows the results of a flow cytometry analysis of cells stainedwith primary antibodies labeled according to methods of the invention.

FIG. 5 shows the results of a flow cytometry analysis of cells stainedwith primary antibodies labeled according to methods of the invention.

FIG. 6 shows images from a microscopic analysis of cells stained withprimary antibodies labeled according to methods of the invention.

FIG. 7 shows images from a microscopic analysis of cells stained withprimary antibodies labeled according to methods of the invention, inaddition to other stains.

FIG. 8 shows the results of a flow cytometry analysis of cells stainedwith primary antibodies labeled according to methods of the invention.

DEFINITIONS

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear, cyclic, or branched. The polymer maycomprise modified amino acids, and it may be interrupted by non-aminoacids. The terms also encompass amino acid polymers that have beenmodified, for example, via sulfonation, glycosylation, lipidation,acetylation, phosphorylation, iodination, methylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, ubiquitination, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” refers to either natural and/or unnatural or synthetic aminoacids, including glycine and both the D or L optical isomers, and aminoacid analogs and peptidomimetics.

A “binding agent” is a molecule that exhibits binding selectivitytowards a binding partner or a target molecule to which it binds. Abinding agent may be a biomolecule such as a polypeptide such as anantibody or protein, polypeptide-based toxin, amino acid, nucleotide,polynucleotides including DNA and RNA, lipids, and carbohydrates, or acombination thereof. A binding agent may also be a hapten, drug,ion-complexing agent such as metal chelators, microparticles, syntheticor natural polymers, cells, viruses, or other fluorescent moleculesincluding the dye molecule according to the invention.

A “targeting moiety” is the portion of the binding agent that binds to abinding partner. A targeting moiety may be, without limitation, anucleotide sequence within a polynucleotide that selectively binds toanother polynucleotide or polypeptide. Another non-limiting example of atargeting moiety may be a polypeptide sequence within a largerpolypeptide sequence which binds specifically to a polynucleotidesequence or a second polypeptide sequence. A targeting moiety may be asmall molecule or structural motif which will bind to a proteinreceptor, another small molecule motif, or complexing agent, withoutlimitation. The selective binding may be a specific binding event.

A “binding partner” is a molecule or particle which is bound by thetargeting moiety. It can be a cell, virus, fragment of a cell, antibody,fragment of an antibody, peptide, protein, polynucleotide, antigen,small molecule, lipid, carbohydrate, or a combination thereof. It may bebound selectively or specifically by the binding agent. In general,binding is considered “specific” when binding between a binding partnerand the target of the binding partner (a binding pair) is at least twotimes greater, such as more than 10, 1000, 1000, 10000 times greater,than background resulting from binding to non-target molecules in asample.

One skilled in the art will realize that such a functional group mayalso be called a reactive group; As used herein, the terms “reactivegroup” and “functional group” refer to chemical groups that can undergochemical reactions with other chemical moieties under certainconditions. When a first reactive group reacts with such a chemicalmoiety (for example a second reactive group), one or more covalent bondsis formed. In such cases, the second reactive group will generally betermed a “functional group”.

As used herein, the term “about” generally means within 10%, 5%, 1%, or0.5% or a given value or range. Alternatively, the term “about” refersto an acceptable standard error of the mean, when considered by one ofordinary skill in the art.

The term “aliphatic” refers to an organic compound or radicalcharacterized by a straight chain or branched chain structure, or closedring structure, any of which contain saturated carbon bonds andoptionally one or more unconjugated carbon-carbon unsaturated bonds,such as a carbon-carbon double bond.

DETAILED DESCRIPTION OF THE INVENTION

In some aspects, the present invention provides methods for labelingtarget proteins with either a reactive dye, a reactive detectable label,or with a reporter protein. The resulting labeled target protein can beused, for example, in subsequent biological staining or otherexperiments. In some embodiments, the resulting labeled target proteinis used without further purification.

In some embodiments, target proteins that are labeled by the methods ofthe invention are members of a binding pair, such as a binding agent anda target. In some embodiments, the binding agent is an antibody and thetarget is an antigen. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain anantigen-binding site which specifically binds (“immunoreacts with”) anantigen. Non-limiting examples include an antibodies, antibodyfragments, recombinant antibodies, non-human antibodies, chimericantibodies, humanized antibodies, or fully human antibodies.Structurally, the simplest naturally occurring antibody (e.g., IgG)comprises four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. The immunoglobulinsrepresent a large family of molecules that include several types ofmolecules, such as IgD, IgG, IgA, IgM and IgE. The term “immunoglobulinmolecule” includes, for example, hybrid antibodies, or alteredantibodies, and fragments thereof. It has been shown that the antigenbinding function of an antibody can be performed by fragments of anaturally-occurring antibody. These fragments are collectively termed“antigen-binding units”. Antigen binding units can be broadly dividedinto “single-chain” (“Sc”) and “non-single-chain” (“Nsc”) types based ontheir molecular structures.

Also encompassed within the terms “antibodies” are immunoglobulinmolecules of a variety of species origins including invertebrates andvertebrates. The term “human” as applies to an antibody or an antigenbinding unit refers to an immunoglobulin molecule expressed by a humangene or fragment thereof. The term “humanized” as applies to a non-human(e.g. rodent or primate) antibodies are hybrid immunoglobulins,immunoglobulin chains or fragments thereof which contain minimalsequence derived from non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, rabbit or primate having thedesired specificity, affinity and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance and minimizeimmunogenicity when introduced into a human body. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody may also comprise atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

Detectable labels, such as amine-reactive detectable labels, aregenerally molecules that can directly or indirectly produce or result ina detectable signal either by themselves or upon interaction withanother molecule. In some embodiments, detectable labels have amolecular weight of less than about 5000 dalton. Non-limiting examplesof detectable labels include labels comprising fluorescent dyes, biotin,digoxin, haptens and epitopes.

Dyes useful in labeling proteins are known in the art. In general, a dyeis a molecule, compound, or substance that can provide an opticallydetectable signal, such as a colorimetric, luminescent, bioluminescent,chemiluminescent, phosphorescent, or fluorescent signal. In someembodiments, the dye is a fluorescent dye. Non-limiting examples ofdyes, some of which are commercially available, include CF dyes(Biotium, Inc.), Alexa Fluor dyes (Invitrogen), DyLight dyes (ThermoFisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.),and HiLyte dyes (Anaspec, Inc.). Other non-limiting example of dyesinclude any of Compounds 1-128, listed in Tables 1-4 and the Examples.In some embodiments, the excitation and/or emission wavelengths of thedye are between 350 nm to 900 nm, or between 400 nm to 700 nm, orbetween 450-650 nm.

In some embodiments, the dye is water soluble. In some embodiments, thedye comprises about, less than about, or more than about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more sulfonate groups. In some embodiments, the dyecomprises about, less than about, or more than about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more water-soluble polymer groups, such as polyethyleneglycol (PEG). A water soluble polymer group can have any suitablemolecular weight, such as 300-10000 Daltons, including 450-5000 Daltons.In some embodiments, a water soluble polymer group has a molecularweight of about, more than about, or less than about 200, 300, 400, 450,500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10000, 20000, 30000, 40000, 50000, or more Daltons, or anyrange between any two such weights. In some embodiments, the dye issulfonated and/or pegylated, such as in a sulfonated and/or pegylatedcoumarin dye, a sulfonated and/or pegylated xanthene dye, a sulfonatedand/or pegylated cyanine dye, or a sulfonated and/or pegylated pyrenedye.

In some embodiments the dye is an amine reactive dye, comprising one ormore groups that react to form an amide bond with an amine. In someembodiments, the amine reactive dye comprises about, less than about, ormore than about 1, 2, or 3 amine-reactive groups. In some embodiments,the amine reactive group is an activated ester. Examples of activatedesters include, but are not limited to, N-hydroxy succinimidyl ester,N-hydroxy sulfosuccinimidyl ester, p-sulfo-tetrafluorophenol ester,pentafluorophenyl esters, tetrafluorophenyl esters, p-nitrophenylesters, 2,4-dinitrophenyl ester, 4-nitrophenyl ester,3-Hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt), carboxylicacids activated using common carbodiimides such as but not limited todiisopropylcarbodiimide (DIPCDI), N,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDC); andcarboxylic acids activated with an uronium salt or a phosphonium salt,such as but not limited to O-Benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU),O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU), 2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU),(2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumtetrafluoroborate) (TCTU),2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU),1-benzotriazolyoxytris-(dimethylamino)phosphonium hexafluorophosphate(BOP) benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PYBOP); or combinations thereof.

Bifunctional crosslinkers for use in the invention generally comprisetwo reactive groups capable of reacting with target and reporterproteins. For example, at least one of the two reactive groups is anamine-reactive group. In some embodiments, a bifunctional crosslinkercomprises an amine-reactive group and a hydrolysis-resistant functionalgroup. The amine-reactive group can react with an amine group within atarget protein, thereby conjugating the crosslinker to the amine groupof the target protein via an amide bond. The amine-reactive group can bean activated ester as described herein. Any of the activated estersdescribed as suitable for use with the amine-reactive dyes oramine-reactive detectable labels are also suitable for use with theamine-reactive group of the crosslinker. As an example, the activatedester may be an N-hydroxy succinimidyl ester.

Hydrolysis-resistant functional groups can react with the reporterprotein, thereby attaching the other end of the crosslinker to thereporter protein. In some embodiments, the functional group ishydrolysis-resistant. For example, a hydrolysis-resistant functionalgroup or hydrolysis-resistant reactive group is at least 90% intact in abuffer, such as the first buffer of the methods of the invention, for atleast 24 hours, or for at least 1 month, or for at least 3 months.Generally, hydrolysis-resistant functional groups are compatible withthe amine-reactive group of the crosslinker such that that they do notreact with each other. Since the amine-reactive group is anelectrophile, the hydrolysis-resistant functional group of thecrosslinker is generally not a nucleophile. Non-limiting examples offunctional groups that are hydrolysis-resistant and non-nucleophilicinclude aldehydes, ketones, azido, alkyne, phosphines participating inStaudinger conjugation, dienes, dienophiles, protected hydrazine andprotected aminooxy. Examples of pairs of hydrolysis-resistant functionalgroups and reactive groups that can react to form a covalent bond arelisted below:

Functional Group Reactive Group Bond formation reaction aldehydeaminooxy oxime formation aldehyde hydrazino hydrazone formation ketoneaminooxy oxime formation ketone hydrazino hydrazone aldehyde acetoneprotected aminooxy oxime formation aldehyde acetone protected hydrazinohydrazone formation ketone acetone protected aminooxy oxime formationketone acetone protected hydrazino hydrazone formation azido alkyneClick chemistry diene dienophile Diels-Alder reaction Azido phosphineStaudinger reaction

In some embodiments, the hydrolysis-resistant functional group excludesmaleimide or disulfide linker-forming groups such as pyridyldisulfide.In one embodiment, the bifunctional crosslinker comprises an N-hydroxysuccinimidyl ester and an aldehyde functional group.

Buffers useful for labeling the target protein with an amine-reactivedye, an amine-reactive detectable label, or with a bifunctionalcrosslinker (i.e., a first reaction buffer) in the methods of theinvention include any that support a labeling reaction as describedherein. In some embodiments, the buffer is an alkaline buffer, having apH greater than about 7, for example having a pH from about 7 to about14, such as a pH of about, more than about 7, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, or 14. In some embodiments the buffer has a pH of about, more thanabout, or less than about 4, 4.5, 5, 5.5, 6, or 6.5. Non-limitingexamples of useful buffers include sodium carbonate buffers, sodiumbicarbonate buffers, borate buffers, tris buffers, MOPS buffers, HEPESbuffers, and combinations thereof. In some embodiments, the buffer is ofa concentration such that the combined solution has a pH from about 6 toabout 11, such as from about 7.9 to about 9.8, or from about 8 to about9. In some embodiments, the pH of the combined solution about, more thanabout, or less than about 6, 6.5, 7, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 10, 10.5, 11, or more. Insome embodiments, the buffer has a starting concentration that is about,more than about, or less than about 2-fold, 5-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, or more than the concentration of the bufferin the combined solution. In one example, a 10× reaction buffercomprises 500 mM sodium bicarbonate and 100 mM Tris.

In some embodiments, the buffer used in the invention comprises a firstamine, for example an aliphatic amine, capable of competing with asecond amine group of the target protein in reacting with the reactivedye or crosslinker described above. In some embodiments, the first amineis a primary or secondary amine, e.g. a primary or secondary aliphaticamine. In general, a first amine is at least 50% less reactive than theω amine of lysine in an amide formation reaction with an activatedcarboxylic acid ester, such as 60%, 70%, 80%, 90%, or less reactive.Reactivity can be measured, for example, as a reaction rate in anaqueous solution. A non-limiting example of a first amine includestris(hydroxymethyl)aminomethane (Tris). In various embodiments, a firstamine with the specified reactivity is a primary or secondary amine. Insome embodiments, the amine acts as a regulating agent of the labelingreaction such that the fraction of labeled to unlabeled amine groups inthe target protein and/or the number of labels per target protein arekept within a desired range. For example, when the amount of antibody isrelatively high, most of the dye, label, or crosslinker molecules reactwith the antibody molecules while a side-reaction between the firstamine and the dye, label, or crosslinker is minimal, such that theantibody is sufficiently labeled. In another example, when the amount oftarget protein (e.g. antibody) is relatively low, the side-reactionbetween the first amine and the dye, label, or crosslinker is relativelyincreased, such that consumption of the dye, label, or crosslinker bythe first amine reduces the likelihood that the small amount of antibodywill be overly labeled. In some embodiments, more than about 95%, 96%,97%, 98%, 99%, 99.5%, or more of the target protein in a sample islabeled. In some embodiments, each target protein is labeled with about,or more than about 1, or with about, more than about, or less thanabout, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 dye, label or crosslinkermolecules. In some embodiments, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,99.5%, or more of labeled target protein in a sample have the samenumber of dye molecules. While not wishing to be bound by theory, it isbelieved that in circumstances where a target protein is mixed with anon-target protein (such as an antibody stabilized with bovine serumalbumin (BSA), and both the target protein and non-target protein arelabeled equally in terms of labeling efficiency per amine basis on eachprotein, a staining process may still tolerate the more or lessequivalent labeling. For example, in one embodiment, non-target proteinmay not have affinity for a subsequent binding target, allowing thenon-target protein to be removed by washing during a subsequent step ofbinding to a biological target.

In various embodiments, a suitable dye is highly water-soluble bypossessing water soluble groups, such as sulfonate group and/or awater-soluble polymer as defined herein. While not wishing to be boundby theory, it is believed that the labeling method can tolerate up to 4to 5 times of a non-target protein (relative to the amount of targetprotein) as described herein. In various embodiments, up to 10 foldnon-target protein may be present without sacrificing the performance ofthe labeled target protein. In general, labeling comprises linking a dyeto a target protein by way of covalent interactions. In someembodiments, labeling comprises formation of a covalent bond, such as anamide bond.

In some embodiments, the amount of target protein in a sample solutionis between about 0.1 μg-10000 μg, such as between 1 μg-1000 μg, 5 μg-200μg, 5 μg-20 μg, 20 μg-50 μg, or 50 μg-100 μg. In some embodiments, theamount of target protein in a sample is about, less than about, or morethan about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,50, 100, 150, 200, 250, 500, 1000, 1500, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, 10000, or more micrograms (μg). In some embodiments,m1 and m2 (both in μg) define the lower and upper limits of an amount oftarget protein in a sample labeling reaction. In some embodiments, therelationship between m1 and m2 for a target protein to be labeled by aspecified amount of reactive dye is expressed as m2/m1, where m2/m1 hasa specified value or range of values. In some embodiments, m1 is fromabout 0.1 μg to about 500 μg, and m2/m1 is from about 1 to about 20. Insome embodiments, m1 is from about 1 μg to about 200 μg, and m2/m1 isfrom about 2 to about 5. In some embodiments, m1 is from about 5 μg toabout 50 μg, and m2/m1 is from about 2 to about 4. In some embodiment,m1 is about 5 μg and m2/m1 is about 4. In some embodiment, m1 is about20 μg and m2/m1 is about 2.5. In some embodiment, m1 is about 50 μg andm2/m1 is about 2.

In some embodiments, the target protein is dissolved in a sample buffer.A target protein dissolved in a sample buffer may have any concentrationsuitable for a labeling reaction, such as about, less than about, ormore than about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or more mg/mL, or any range therebetween, such as from about0.01-10 mg/mL, 0.1-5 mg/mL, or 0.5-1 mg/mL. A buffer for dissolving atarget protein, such as an antibody, may be any of a number of commonlyused biological buffers, including but not limited to phosphate bufferedsaline (PBS), Tris, MOPS, HEPES, and combinations thereof. In someembodiments, the concentration of Tris is less than about 1000 mM, 100mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 5 mM, or less.

In some embodiments, a sample solution comprising a target protein alsocomprises an amount of one or more stabilizers and/or one or morepreservatives. Common stabilizers include, but are not limited to,bovine serum albumin (BSA), gelatin and glycerol. In some embodiments,the amount of BSA or gelatin is about, more than about, or less thanabout 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 9-fold, 8-fold,7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1-fold, or less than theamount of the target protein by weight. In some embodiments, the amountof glycerol is about, more than about, or less than about 50%, 25%, 20%,15%, 10%, 5%, 4%, 3%, 2%, 1% or less of the buffer. Common preservativesinclude, but are not limited to, sodium azide, thimerosal, and otherantimicrobial agents. A sample solution may or may not be pretreated topurify a target protein away from one or more other sample solutioncomponents. In some embodiments, the sample solution is not pretreatedto purify the target protein away from one or more other components ofthe sample solution. Methods for removing one or more excess samplesolution components, such as preservatives and/or stabilizers, are knownin the art, non-limiting examples of which include protein A-basedaffinity chromatography, size-exclusion chromatography, and ultramembrane filtration. Commercial kits for protein purification, such asantibody cleaning kits, may also be used for removing one or more excesssample solution components. Size-exclusion column chromatography andultra membrane filtration are useful for removingstabilizers/preservatives of relatively small molecules, such asglycerol, Tris, and amino acids. Ultra membrane filtration may also beused to adjust target protein concentration to a desired value. Ultramembrane filtration can be conveniently and rapidly carried out on acentrifuge using a commercial ultra membrane filtration vial. Themembranes in such ultra filtration devices typically have different poresizes, or so-called Molecular Weight Cut-off sizes (MWCO), permittingrelatively small molecules to go through while retaining biggermolecules, such as proteins. In some embodiments, a membrane with a MWCOof about, less than about, or more than about 1, 5, 10, 15, 20, 25, 50,100, or more kD is used to purify a target protein before labeling.Purification methods may be used to purify labeled protein.

In some embodiments, the labeling reaction with a dye, detectable labelor crosslinker takes place at temperature of about, less than about, ormore than about 4° C., 10° C., 15° C., 16° C., 17° C., 18° C., 19° C.,20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C.,29° C., 30° C., 37° C., 40° C., 50° C., or higher. In some embodiments,the labeling reaction takes place a room temperature, such as between15° C.-30° C. In some embodiments, the labeling reaction is completed inless than about 5, 4, 3, 2, 1, or fewer hours; or less than about 60,45, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, or fewer minutes. In someembodiments a completed labeling reaction containing a target proteinlabeled with a detectable label such as a dye, is used, such as in adetection process, without further manipulation, such as without theaddition of other reagents, such as a quenching agent, and/or withoutpurification, isolation, or concentration of the labeled target protein.Likewise, in some embodiments, a completed labeling reaction containinga target protein labeled with a crosslinker can be used directly in asubsequent protein-protein conjugation reaction without any purificationof the crosslinker-labeled target protein as described herein.

A second reaction buffer used in the protein-protein conjugation methodof the invention can be any buffer that facilitates the reaction betweenthe hydrolysis-resistant functional group of the crosslinker and thepairing hydrolysis-resistant reactive group on the reporter proteindescribed further in details below. In some embodiments, the secondbuffer comprises a catalyst that can accelerate the conjugationreaction. Similar to the reaction buffer used in detectable label-targetprotein conjugation or the first reaction buffer used in conjugating thecrosslinker to the target protein, in some embodiments the secondreaction buffer is provided as a concentrated solution that is about,more than about, or less than about 2-fold, 5-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, or more than the concentration of the bufferin the second combined solution. In some embodiments, the second bufferis a buffer that facilities an oxime or hydrazone bond formation, suchas the reaction between an aldehyde functional group and an aminooxy orhydrazino reactive group. Oxime and hydrazone formation generallyprefers a relatively acidic buffer, such as a buffer with a pH fromabout 3 to about 7. Generally, aryl amines, such as aniline, are knownto catalyze oxime and hydrazone formation. Thus, according to oneembodiment, the second buffer is a phosphate buffer with a pH betweenabout 3 and about 4, further comprising between about 1 and about 1000,or about 10 and about 200, or about 50 and about 150, or about 75 andabout 125 mM aniline. In some embodiments, the second buffer comprisesbetween about 95 and 105 mM aniline, for example about 100 mM aniline.

A reporter protein suitable for the protein-protein conjugation methodis generally a protein that can produce a detectable signal either byitself or upon interaction with another molecule. According to oneembodiment, reporter proteins are enzymes that upon interaction with acolorogenic or fluorogenic enzyme substrate produce a colored orfluorescent product. For example, horseradish peroxidase (HRP) andalkaline phosphatase (AP) both are commonly used as reporter proteinsthat interact with a substrate to produce a detectable colored orfluorescent product. Some enzyme reporter proteins can also interactwith a substrate to produce detectable luminescence or bioluminescence.According to one embodiment, reporter proteins are fluorescent proteins,which are natural or recombinant protein molecules that bear one or morefluorophores. According to one preferred embodiment, fluorescentproteins are phycobiliproteins. Phycobiliproteins are a family offluorescent proteins found in cyanobacteria and eukaryotic red algae. Innature, these highly colored proteins function as light-harvestingantenna molecules to collect photo energy for biochemical synthesis.Phycobiliproteins are considered to be the brightest fluorescent dyesdue to their large extinction coefficient (700,000-2,410,000) and highfluorescence quantum yield (0.68-0.98). As a result, phycobiliproteinsare widely used as fluorescent labels for biological detection, offeringsensitivity many times higher than small synthetic dyes.Phycobiliprotein-labeled probes are extensively used in bead-baseddetection, microarrays and flow cytometry. In some preferredembodiments, phycobiliproteins are R-phycerythrine (R-PE),allophycocianin (APC) and Peridinin-chlorophyll-protein (PerCP). R-PEand PerCP are both excitable by the common 488 nm argon laser, but emitat ˜578 nm and ˜675 nm, respectively. APC, on the other hand, isexcitable by the red He—Ne laser with emission at ˜660 nm. The varietyof emission wavelengths by phycobiliproteins can be further extended bythe use of so-called tandem dyes. Tandem dyes are energy transfer dyesprepared by covalently labeling phycobiliproteins with a suitablesynthetic dye that acts as energy receptor. In a tandem dye, thephycobiliprotein acts an antenna dye to efficiently absorb excitationlight and then transfer its emission energy to the acceptor syntheticdye, which re-emits at the emission wavelength of the acceptor dye. Byusing appropriate acceptor dyes, the original emission wavelengths ofphycobiliproteins can be extended to virtually anywhere between about580 nm and 800 nm. Thus, by using a combination of regular syntheticdyes, phycobiliproteins and tandem dyes, it is possible for one todetect many biological targets in the same sample by using only alimited number of excitation sources. For example, by using acombination of dyes, it is possible to detect over 10 targets in thesame sample by flow cytometry.

In some embodiments of the methods of the invention, the reporterproteins are pre-functionalized with a hydrolysis-resistant reactivegroup capable of reacting with the hydrolysis-resistant functional groupof the crosslinker-modified target protein to form a covalent linkage.The pre-functionalized reporter protein is usually purified such that itis substantially free of any unattached small molecule comprising thesame reactive group that may interfere with the protein-proteinconjugation reaction. The pre-functionalized reporter protein may beprepared by reacting any of the above mentioned reporter protein with acrosslinker comprising a first reactive group capable of reacting withan amine or thiol on the reporter protein to form a covalent linkage anda second hydrolysis-resistant reactive group capable of reacting withthe functional group on the crosslinker-modified target protein. Thefunctionalization reaction of the reporter protein may be carried out inany of the buffers mentioned previously herein. The functionalizedreporter protein may be purified by size exclusion column, ultramembranefiltration or dialysis to remove any unattached crosslinker. In somecases, the hydrolysis-resistant reactive group may not be a pairingreactive group to the hydrolysis-resistant functional group on thecrosslinker-modified target protein; that is, the initialhydrolysis-resistant reactive group on the reporter protein may notreact with the hydrolysis-resistant functional group on the targetprotein. In such a case, the initially functionalized reporter proteinis usually reacted with a second crosslinker which, upon reacting withthe initially functionalized reporter protein, provides a suitablehydrolysis-resistant reactive group for conjugation to the targetprotein.

According to one embodiment, the invention provides a reporter proteinprefunctionalized with an aminooxy or hydrazino group, wherein thereporter protein comprises 1 to about 15 aminooxy or hydrazino groupsand the wherein the functionalized reporter protein is purified toremove unreacted aminooxy or hydrazino crosslinker. The aminooxy orhydrazino groups herein are usually “free” reactive group, i.e. they arenot in a protected form, such as aminooxy or hydrazino groups protectedby acetone in the form of an oxime or hydrazone. Free aminooxy orhydrazino groups possess much higher reactivity toward aldehyde orketone groups in conjugation reaction than their protected forms. In oneembodiment, the reporter protein comprises 1 to about 6 aminooxy orhydrazino groups. According to some embodiments, the pre-functionalizedreporter proteins are aminooxy-functionalized reporter proteins selectedfrom the list consisting of phycobiliproteins, tandem dyes, horseradishperoxidase and alkaline phosphatase. In some embodiments, thefunctionalized reporter protein may be provided as lyophilized solid oras a solution.

Provided herein is also a method of preparing an aminooxy- orhydrazino-functionalized reporter protein, comprising the steps of: (a)reacting a reporter protein with a bifunctional crosslinker comprisingan amine-reactive group and an aldehyde or ketone group in a buffer fora time sufficient to form an aldehyde or ketone functionalized reporterprotein; (b) reacting the aldehyde- or ketone-functionalized reporterprotein with an excess of a bis-aminooxy, a bis-hydrazino or aaminooxy-hydrazino crosslinker in a buffer for a time sufficient to forman aminooxy- or hydrazino-functionalized reporter protein; and (c)purifying said functionalized reporter protein.

The conjugation of a reporter protein to the crosslinker-modified targetprotein can be carried out at any temperature from above 0° C. to about90° C., depending on the stability of the protein. More typically, thereaction is carried out from about 4° C. to about 45° C. In someembodiments, the reaction is carried out at room temperature, such asfrom about 15° C. to about 30° C. The reaction time may be longer thanthat for functionalizing the target protein with the crosslinker becauseof the relatively large size of the two proteins. When the reaction iscarried out at room temperature, the reaction time may be greater than15 minutes, or greater than 30 minutes. In some embodiments, thereaction time is about 1 hour at room temperature. In some embodiments acompleted labeling reaction containing a target protein labeled with areporter protein is used, such as in a detection process, withoutfurther manipulation, such as without the addition of other reagents,such as a quenching agent, and/or without purification, isolation, orconcentration of the labeled target protein.

In some embodiments, one or more portions of an amount of labeledprotein produced by the labeling reaction are used directly in one ormore processes, and unused labeled protein is stored for later use. Insome embodiments, a storage buffer is added to a labeled protein toproduce a stored protein solution. In general, a storage buffer iseffective in increasing the time following a labeling reaction duringwhich a labeled protein remains stable. In general, a labeled protein isconsidered “stable” when it retains its activity, or a substantialportion thereof, such as for use in a detection process. In someembodiments, a stored protein is considered stable when about or morethan about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97.5%, 99%,99.5%, 99.9%, or more of the pre-storage activity remains. In someembodiments, the stored labeled protein is stable for about or more thanabout 1, 2, 3, 4, 5, 6, 7, 8, or more weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or more months; or 1, 2, 3, or more years. In someembodiments, a storage buffer comprises one or more stabilizers and/orone or more preservatives. Non-limiting examples of stabilizers includebovine serum albumin, gelatin, and glycerol. Non-limiting examples ofpreservatives include sodium azide, thimerosal, and other antimicrobialagents. In general, a storage buffer can have either a lower pH orhigher pH than the buffer used in the labeling reaction, such thataddition of the storage buffer lowers or raises the pH of the combinedsolution containing the labeled protein. In some embodiments, the pH ofa solution containing a stored, labeled protein is about, less thanabout, or more than about 5.5, 6, 6.5, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.5, 9, or higher. In some embodiments, the pH of thestored protein solution is between about 6.0-9.0, such as 6.5-8.0, or7.2-7.5.

Labeled proteins of the present invention can be used as indicators ofthe presence, absence, relative abundance, and/or amount of a biologicaltarget, such as may be found in a biological sample or organism, forexample by use in a detection process. In one aspect, the inventionprovides a method for staining one or more biological targets. In oneembodiment, the method comprises (a) preparing one or more labeledproteins according to a method of the present invention, wherein each ofsaid one or more labeled proteins comprises a targeting moiety thatbinds to a binding partner associated with one or more of saidbiological targets; and, (b) exposing said one or more biologicaltargets to said one or more labeled proteins, such that said labeledprotein binds to said binding partner thereby staining said one or morebiological targets. In some embodiments, step (b) is performed withoutpurification of the one or more labeled proteins. In some embodiments,step (a) is not terminated by the addition of a quencher.

In general, a biological sample refers to any biological tissue orfluid. In some embodiments, samples include, but are not limited to,cells in culture, bone marrow; blood; blood cells (e.g., white bloodcells, red blood cells, etc.); ascites; tissue or fine needle biopsysamples; cell-containing body fluids; free floating nucleic acids;sputum; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid;washings or lavages such as a ductal lavages or broncheoalveolarlavages; aspirates; scrapings; bone marrow specimens; tissue biopsyspecimens; surgical specimens; other body fluids, secretions, and/orexcretions; and/or cells therefrom. In some embodiments, a samplecomprises cells obtained from a patient. The cells may be, for example,from blood, bone marrow, and/or from tissue derived from solid organs,such as brain, spleen, bone, heart, vascular, lung, kidney, liver,pituitary, endocrine glands, lymph node, dispersed primary cells, tumorcells. Biological samples may include sections of tissues, including butnot limited to frozen or fixed sections taken for histological purposes.In some embodiments, a sample may be a body fluid, including, but notlimited to, blood fluids, lymph, ascitic fluids, gynecological fluids,and urine. Samples may be obtained from a subject by any of a widevariety of methods known in the art, including without limitation biopsy(e.g., fine needle aspiration or tissue biopsy), surgery, and collectionof body fluid (e.g., blood, lymph, etc.). Biological samples alsoinclude any material derived by processing any of the above samples.Derived samples may, for example, include nucleic acids or proteinsextracted from the biological sample, or obtained by subjecting thesample to techniques such as amplification or reverse transcription ofmRNA, or isolation and/or purification of certain components.

A wide variety of cell lines for tissue culture are known in the art.Examples of cell lines include, but are not limited to, C8161, CCRF-CEM,MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn,HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10,T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2,P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1,BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRCS, MEF, Hep G2, HeLa B,HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial,BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetalfibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780,A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36,Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr−/−, COR-L23,COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT,CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0,FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60,HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1,LNCap, Ma-MeI 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468,MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd,NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1,NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassus, Va.)).

In some embodiments, staining of the one or more biological target isperformed in vivo or in vitro. Many methods for staining or otherwisedetecting a biological target using binding partners are known in theart, and include without limitation immunohistochemistry, immunosorbentassays (e.g. ELISA, with or without a linked enzyme), flow cytometry,fluorescence activated cell sorting (FACS), microarray (e.g. proteinarray), and other binding partner assays whereby the presence of abinding partner is indicated by retention by an organism, sample, orportion thereof of the one or more labeled protein (e.g. Western blot).In some embodiments, the label carried by the labeled protein isdirectly observable, such as in a fluorescent dye detectable by exposureto light of a particular frequency or range of frequencies. Typically,excess labeled protein is washed away prior to detection, such thatlabeled protein that remains is indicative of the presence, absence,relative abundance, and/or quantity of the biological target. Examplesof biological targets include, but are not limited to, amino acids,polypeptides, nucleotides, polynucleotides (e.g. DNA or RNA),carbohydrates, lipids, metabolites, cell signaling molecules, cluster ofdifferentiation proteins, hormones, cell surface proteins, intracellularproteins, fragments thereof, and combinations or complexes thereof. Insome embodiments, two or more different labeled proteins, eachrecognizing a different binding partner or set of binding partners areused to detect two or more different biological targets in a singlesample or organism. The different labeled proteins typically comprisedifferent targeting moieties, and may or may not comprise the same dye.In some embodiments, each of two or more different labeled proteins arelabeled with different dyes, such that exposing two or more biologicaltargets to the two or more differently labeled proteins renders the twoor more biological targets optically distinguishable. In someembodiments, one or more biological targets are exposed to a primarybinding agent, such as an antibody, that is unlabeled, before exposureto a labeled protein that specifically binds the primary binding agent.

In some embodiments, stained biological targets are analyzed by flowcytometry. Methods and devices for carrying out flow cytometry on cellsand other particles are known in the art. Flow cytometry is a techniquefor counting, examining, and/or sorting microscopic particles suspendedin a stream of fluid. It allows simultaneous multiparametric analysis ofthe physical and/or chemical characteristics of single cells flowingthrough an optical/electronic detection apparatus. A description of atypical flow cytometry process follows. One or more beams of light,usually laser light, of one or more frequencies are directed onto ahydrodynamically focused stream of fluid. A number of detectors areaimed at the point where the stream passes through the light beam; onein line with the light beam (Forward Scatter or FSC) and severalperpendicular to it (Side Scatter (SSC) and one or more fluorescentdetectors. Each suspended particle passing through the beam scatters thelight in some way, and fluorescent chemicals in the particle may beexcited into emitting light at a lower frequency than the light source.This combination of scattered and fluorescent light is picked up by thedetectors, and by analyzing fluctuations in brightness at each detector(one for each fluorescent emission peak), it is possible to deducevarious facts about the physical and chemical structure of eachindividual particle. FSC correlates with the cell size and SSC dependson the inner complexity of the particle, such as shape of the nucleus,the amount and type of cytoplasmic granules or the membrane roughness.Subsequent to detection, cells may be routed to a receptacle based onone or more characteristics measured by said detection so as to sort andphysically group cells based on such characteristics, such as influorescence activated cell sorting (FACS). Each of a plurality ofreceptacles may be designated to contain a different set of cells, eachset having a different characteristic or set of characteristics.Receptacles can be any suitable container, including but not limited towells of a multi-well plate; tubes; and ordered tubes, such as in a rackor an array.

In some embodiments, stained biological targets are analyzed by westernblot. Methods and systems for carrying out Western blots are known inthe art. Briefly, this method involves separation of a substrate fromother protein by means of a separation medium, such as an acrylamidegel, followed by transfer of the substrate to a membrane (e.g., nylon orPVDF). Presence of the substrate is then detected by labeled bindingpartners specific for the substrate, such as antibodies, which may inturn be detected by a further binding reagent. Where this first bindingpartner is a labeled protein, use of further binding reagents may not benecessary. This method enables both quantitation of an amount ofsubstrate and determination of its identity by a relative position onthe membrane which is indicative of a migration distance in theseparation medium (e.g. during electrophoresis).

In some embodiments, stained biological targets are analyzed byimmunohistochemistry. Methods and systems for immunohistochemicalanalysis are known in the art. In general, detections involving cellsinclude sample fixation and permeabilization, sample blocking with ablocking solution, incubation of the sample with one or more labeledprimary binding agents, washing of the stained sample, and detection.Typically, sections of a tissue sample are adhered to a microscopeslide. The sample is then exposed to one or more binding agents, eachwith specificity for one or more targets. Where the one or more bindingagents comprise a detectable label, such as a dye, staining is evaluatedby detecting the detectable label. Where the one or more binding agentsdo not comprises a detectable label, the sample are exposed to one ormore second binding agents, each with specificity to one or more of thefirst binding agents and comprising a detectable label, such as a dye.In some embodiments, stained samples are analyzed under a microscope.The combination of exposure to unlabeled binding agent followed byexposure to a second, labeled binding agent can be used in combinationwith any detection process, for example to amplify a signal.

In some embodiments, the method further comprises visualizingfluorescence of the stained one or more biological targets.Visualization may be by eye, or may be performed by a device, such as acamera, whereby an image is produced. Typically, visualization isperformed after a washing step to remove excess labeled protein. In someembodiments, visualization is performed immediately after washing. Insome embodiments, visualization is performed at about, before about, orafter about 1, 5, 10, 15, 20, 30, 45, 60, 90, 180, or more minutes afterwashing; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 24, or more hours afterwashing; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 60, or moredays after washing. In some embodiments, a biological samples stained bythe methods of the invention are stored for later visualization. Ingeneral, visualization involves detecting the presence and optionallylevel of one or more dyes used to stain one or more biological targetsin a biological sample, which in turn is indicative of the presence andoptionally level of the one or more biological targets in a biologicalsample. Where the dye is fluorescent dye, visualization may compriseexposure to an excitation frequency, following by detecting frequencyand/or intensity of fluorescent light emitted by one or more dyes usedto stain a biological sample. In general, the wavelength of excitationfor a given dye is shorter than the wavelength emitted by the dye. Insome embodiments, the excitation wavelength is about, less than about,or more than about 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 615, 620 nm, 625,630 nm, 635, 640 nm, 645, 650 nm, 655, 660 nm, 665, 670 nm, 675, 680 nm,685, 690 nm, 695, 700 nm, 705, 710 nm, 715, 720 nm, 725, 730 nm, 735,740 nm, 745, 750 nm, 755, 760 nm, 765, 770 nm, 775, 780 nm, 790 nm, 800nm, 810 nm, 820 nm, 830 nm, 840 nm, 850 nm, 860 nm, 870 nm, 880 nm, 890nm, 900 nm, 910 nm, 920 nm, 930 nm, 940 nm, 950 nm, 960 nm, 970 nm, 980nm, 990 nm, 1000 nm, 1020 nm, 1040 nm, 1060 nm, 1080 nm, 1100 nm, 1120nm, 1140 nm, 1160 nm, 1180 nm, 1200 nm, or any range including two suchwavelengths as endpoints, such as from about 350 nm to about 1200 nm,and from about 450 nm to about 750 nm. In some embodiments, the emissionwavelength is about, less than about, or more than about 250 nm, 300 nm,350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm,600 nm, 610 nm, 615, 620 nm, 625, 630 nm, 635, 640 nm, 645, 650 nm, 655,660 nm, 665, 670 nm, 675, 680 nm, 685, 690 nm, 695, 700 nm, 705, 710 nm,715, 720 nm, 725, 730 nm, 735, 740 nm, 745, 750 nm, 755, 760 nm, 765,770 nm, 775, 780 nm, 790 nm, 800 nm, 810 nm, 820 nm, 830 nm, 840 nm, 850nm, 860 nm, 870 nm, 880 nm, 890 nm, 900 nm, 910 nm, 920 nm, 930 nm, 940nm, 950 nm, 960 nm, 970 nm, 980 nm, 990 nm, 1000 nm, 1020 nm, 1040 nm,1060 nm, 1080 nm, 1100 nm, 1120 nm, 1140 nm, 1160 nm, 1180 nm, 1200 nm,1220 nm, 1240 nm, 1250 nm, or any range including two such wavelengthsas endpoints, such as from about 360 nm to about 1250 nm.

In some embodiments, a diagnosis of a condition in a subject is madebased on the results of the staining process. Conditions that may bediagnosed according to these methods include any that are associatedwith a detectable marker or characteristic that can be detected byexposure to a labeled protein of the present invention. For example, alarge number of antibodies against various disease targets, includingbut not limited to tumor-associated antigens, have been deposited at theAmerican Type Culture Collection (ATCC) and/or have published variableregion sequences and are available for use in the claimed methods andcompositions. Antibody sequences or antibody-secreting hybridomasagainst almost any disease-associated antigen may be obtained by asimple search of the ATCC, NCBI and/or USPTO databases for antibodiesagainst a selected disease-associated target of interest. The antigenbinding domains of the cloned antibodies may be amplified, excised,ligated into an expression vector, transfected into an adapted host celland used for protein production, using standard techniques well known inthe art. Examples of diseases and conditions that may be identifiedusing the methods and compositions of the invention include, but are notlimited to, inherited diseases, infectious diseases, diseases arisingfrom genetic mutation, poisoning, and cancer.

In one aspect, the invention provides a method for evaluating theefficacy of a test compound. In one embodiment, the method comprises (a)exposing a system comprising a drug target to a test compound; (b)staining one or more biological targets in said system according to amethod of the invention; and evaluating the efficacy of said testcompound based on the results of step (b). The drug target and thebiological target may be the same or different. In general, efficacy ismeasured by comparing the degree of an effect in a system receiving thetest compound with the degree of an effect in a system receiving nocompound or a reference compound. In general, an effect is measured bydetecting the stained biological target, such as by visualizing afluorescent dye. Examples of effects on biological targets as may bedetected using a labeled protein of the invention include, but are notlimited to, changes in absolute abundance, changes in relativeabundance, changes in localization (such as within a cell, within atissue, within an organ, or within an organ system), and any effect thatcan be observed or quantified by visualizing the labeled protein.Systems that can be exposed to a drug target include in vitro systemsand in vivo systems, such as reactions in solution, cultured cells, andwhole organisms. Staining may be performed on the system itself, or on asample therefrom. For example, a test compound may be delivered to asubject, such as a human, and an effect on a tissue of the subject maybe evaluated in a sample of the tissue removed from the subject bystaining the sample according to the methods of the invention.

Examples of test compounds include, but are not limited to, drugs, smallmolecules, activators, inhibitors, antitumor agents, anti-miotics,steroids, sympathomimetics, local anesthetics, antimicrobial agents,antihypertensive agents, antihypertensive diuretics, cardiotonics,coronary vasodilators, vasoconstrictors, β-blockers, antiarrhythmicagents, calcium antagonists, anti-convulsants, agents for dizziness,tranquilizers, antipsychotics, muscle relaxants, respiratory agents,non-steroidal hormones, antihormones, vitamins, herb medicines,antimuscarinic, muscarinic cholinergic blocking agents, mydriatics,psychic energizers, humoral agents, antispasmodics, antidepressantdrugs, anti-diabetics, anorectic drugs, anti-allergenics, decongestants,antipyretics, antimigrane, anti-malarials, anti-ulcerative, peptides,anti-estrogen, anti-hormone agents, antiulcer agents, anesthetic agent,drugs having an action on the central nervous system, growth factors,mitogens, cytokines, adhesion molecules, hormones, polynucleotides,antibodies, natural compounds, lactones, chemotherapeutic agents, immunemodulators, carbohydrates, proteases, ions, reactive oxygen species,protein fragments, or combinations thereof. In some embodiments, aphysical parameter is used to modulate the system instead of a testcompound having a drug target. Examples of physical parameters include,but are not limited to radiation, heat, cold, UV radiation, changes inpressure, and combinations thereof.

The test compounds may be found and/or isolated from a variety of customand commercially available combinatorial libraries. In one embodiment,the pool of test compounds may include libraries of antitumor agentssuch as, chemotherapeutic agent is selected from the group consisting ofadriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin,actinomycin-D, mitomycin C, cisplatin (CDDP), doxorubicin, etoposide,verapamil, podophyllotoxin, carboplatin, procarbazine, mechlorethamine,cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan,nitrosurea, dactinomycin, daunorubicin, bleomycin, plicomycin,mitomycin, tamoxifen, taxol, transplatinum, vincristin, vinblastin,methotrexate, pilocarpine, mixtures and combinations thereof and thelike.

Analgesic anti-inflammatory agents such as, acetaminophen, aspirin,salicylic acid, methyl salicylate, choline salicylate, glycolsalicylate, 1-menthol, camphor, mefenamic acid, fluphenamic acid,indomethacin, diclofenac, alclofenac, ibuprofen, ketoprofen, naproxene,pranoprofen, fenoprofen, sulindac, fenbufen, clidanac, flurbiprofen,indoprofen, protizidic acid, fentiazac, tolmetin, tiaprofenic acid,bendazac, bufexamac, piroxicam, phenylbutazone, oxyphenbutazone,clofezone, pentazocine, mepirizole, and the like. Drugs having an actionon the central nervous system, for example sedatives, hypnotics,antianxiety agents, analgesics and anesthetics, such as, chloral,buprenorphine, naloxone, haloperidol, fluphenazine, pentobarbital,phenobarbital, secobarbital, amobarbital, cyclobarbital, codeine,lidocaine, tetracaine, dyclonine, dibucaine, cocaine, procaine,mepivacaine, bupivacaine, etidocaine, prilocalne, benzocaine, fentanyl,nicotine, and the like. Local anesthetics such as, benzocaine, procaine,dibucaine, lidocaine, and the like.

Antihistaminics or antiallergic agents such as, diphenhydramine,dimenhydrinate, perphenazine, triprolidine, pyrilamine, chlorcyclizine,promethazine, carbinoxamine, tripelennamine, brompheniramine,hydroxyzine, cyclizine, meclizine, clorprenaline, terfenadine,chlorpheniramine, and the like. Anti-allergenics such as, antazoline,methapyrilene, chlorpheniramine, pyrilamine, pheniramine, and the like.Decongestants such as, phenylephrine, ephedrine, naphazoline,tetrahydrozoline, and the like.

Antipyretics such as, aspirin, salicylamide, non-steroidalanti-inflammatory agents, and the like. Antimigrane agents such as,dihydroergotamine, pizotyline, and the like. Acetonide anti-inflammatoryagents, such as hydrocortisone, cortisone, dexamethasone, fluocinolone,triamcinolone, medrysone, prednisolone, flurandrenolide, prednisone,halcinonide, methylprednisolone, fludrocortisone, corticosterone,paramethasone, betamethasone, ibuprophen, naproxen, fenoprofen,fenbufen, flurbiprofen, indoprofen, ketoprofen, suprofen, indomethacin,piroxicam, aspirin, salicylic acid, diflunisal, methyl salicylate,phenylbutazone, sulindac, mefenamic acid, meclofenamate sodium,tolmetin, and the like. Muscle relaxants such as, tolperisone, baclofen,dantrolene sodium, cyclobenzaprine.

Steroids such as, androgenic steroids, such as, testosterone,methyltestosterone, fluoxymesterone, estrogens such as, conjugatedestrogens, esterified estrogens, estropipate, 17-β estradiol, 17-βestradiol valerate, equilin, mestranol, estrone, estriol, 17β ethinylestradiol, diethylstilbestrol, progestational agents, such as,progesterone, 19-norprogesterone, norethindrone, norethindrone acetate,melengestrol, chlormadinone, ethisterone, medroxyprogesterone acetate,hydroxyprogesterone caproate, ethynodiol diacetate, norethynodrel, 17-αhydroxyprogesterone, dydrogesterone, dimethisterone, ethinylestrenol,norgestrel, demegestone, promegestone, megestrol acetate, and the like.

Respiratory agents such as, theophilline and β 2-adrenergic agonists,such as, albuterol, terbutaline, metaproterenol, ritodrine, carbuterol,fenoterol, quinterenol, rimiterol, solmefamol, soterenol, tetroquinol,and the like. Sympathomimetics such as, dopamine, norepinephrine,phenylpropanolamine, phenylephrine, pseudoephedrine, amphetamine,propylhexedrine, arecoline, and the like.

Antimicrobial agents including antibacterial agents, antifungal agents,antimycotic agents and antiviral agents; tetracyclines such as,oxytetracycline, penicillins, such as, ampicillin, cephalosporins suchas, cefalotin, aminoglycosides, such as, kanamycin, macrolides such as,erythromycin, chloramphenicol, iodides, nitrofrantoin, nystatin,amphotericin, fradiomycin, sulfonamides, purrolnitrin, clotrimazole,miconazole chloramphenicol, sulfacetamide, sulfamethazine, sulfadiazine,sulfamerazine, sulfamethizole and sulfisoxazole; antivirals, includingidoxuridine; clarithromycin; and other anti-infectives includingnitrofurazone, and the like.

Antihypertensive agents such as, clonidine, α-methyldopa, reserpine,syrosingopine, rescinnamine, cinnarizine, hydrazine, prazosin, and thelike. Antihypertensive diuretics such as, chlorothiazide,hydrochlorothrazide, bendoflumethazide, trichlormethiazide, furosemide,tripamide, methylclothiazide, penfluzide, hydrothiazide, spironolactone,metolazone, and the like. Cardiotonics such as, digitalis,ubidecarenone, dopamine, and the like. Coronary vasodilators such as,organic nitrates such as, nitroglycerine, isosorbitol dinitrate,erythritol tetranitrate, and pentaerythritol tetranitrate, dipyridamole,dilazep, trapidil, trimetazidine, and the like. Vasoconstrictors suchas, dihydroergotamine, dihydroergotoxine, and the like. β-blockers orantiarrhythmic agents such as, timolol pindolol, propranolol, and thelike. Humoral agents such as, the prostaglandins, natural and synthetic,for example PGE 1, PGE 2 α, and PGF 2 α, and the PGE 1 analogmisoprostol. Antispasmodics such as, atropine, methantheline, papavenne,cinnamedrine, methscopolamine, and the like.

Calcium antagonists and other circulatory organ agents, such as,aptopril, diltiazem, nifedipine, nicardipine, verapamil, bencyclane,ifenprodil tartarate, molsidomine, clonidine, prazosin, and the like.Anti-convulsants such as, nitrazepam, meprobamate, phenyloin, and thelike. Agents for dizziness such as, isoprenaline, betahistine,scopolamine, and the like. Tranquilizers such as, reserprine,chlorpromazine, and antianxiety benzodiazepines such as, alprazolam,chlordiazepoxide, clorazeptate, halazepam, oxazepam, prazepam,clonazepam, flurazepam, triazolam, lorazepam, diazepam, and the like.Antipsychotics such as, phenothiazines including thiopropazate,chlorpromazine, triflupromazine, mesoridazine, piperracetazine,thioridazine, acetophenazine, fluphenazine, perphenazine,trifluoperazine, and other major tranqulizers such as, chlorprathixene,thiothixene, haloperidol, bromperidol, loxapine, and molindone, as wellas, those agents used at lower doses in the treatment of nausea,vomiting, and the like.

Respiratory agents such as, codeine, ephedrine, isoproterenol,dextromethorphan, orciprenaline, ipratropium bromide, cromglycic acid,and the like. Non-steroidal hormones or antihormones such as,corticotropin, oxytocin, vasopressin, salivary hormone, thyroid hormone,adrenal hormone, kallikrein, insulin, oxendolone, and the like. Vitaminssuch as, vitamins A, B, C, D, E and K and derivatives thereof,calciferols, mecobalamin, and the like for dermatologically use. Enzymessuch as, lysozyme, urokinaze, and the like. Herb medicines or crudeextracts such as, Aloe vera, and the like.

Mydriatics such as, atropine, cyclopentolate, homatropine, scopolamine,tropicamide, eucatropine, hydroxyamphetamine, and the like. Psychicenergizers such as 3-(2-aminopropy)indole, 3-(2-aminobutyl)indole, andthe like. Antidepressant drugs such as, isocarboxazid, phenelzine,tranylcypromine, imipramine, amitriptyline, trimipramine, doxepin,desipramine, nortriptyline, protriptyline, amoxapine, maprotiline,trazodone, and the like.

Anti-diabetics such as, insulin, and the like and anticancer drugs suchas, tamoxifen, methotrexate, and the like. Anorectic drugs such as,dextroamphetamine, methamphetamine, phenylpropanolamine, fenfluramine,diethylpropion, mazindol, phentermine, and the like. Anti-malarials suchas, the 4-aminoquinolines, alphaminoquinolines, chloroquine,pyrimethamine, and the like. Anti-ulcerative agents such as,misoprostol, omeprazole, enprostil, and the like. Antiulcer agents suchas, allantoin, aldioxa, alcloxa, N-methylscopolamine methylsuflate, andthe like. The drugs mentioned above may be used in combination asrequired. Moreover, the above drugs may be used either in the free formor, if capable of forming salts, in the form of a salt with a suitableacid or base. If the drugs have a carboxyl group, their esters may beemployed.

In general, a biological target is any target comprising a bindingpartner to which a labeled protein of the current invention specificallybinds. The methods and compositions of the invention may be employed toexamine and profile the status of any biological target in a cellularpathway, or collections of such biological targets. Single or multipledistinct pathways may be profiled (sequentially or simultaneously), orsubsets of biological targets within a single pathway or across multiplepathways may be examined (again, sequentially or simultaneously).

A wide variety of activation events can be detected by labeled proteinsof the present invention. In general, the basic requirement is that thechange in activation results in a change in the biological target thatis detectable by altered binding to a labeled protein. What is importantis to differentiate, using a labeled protein that specifically binds toa binding partner that is indicative of an activation state, thusdistinguishing between two or more activation states (e.g. “off” and“on”). Typically, the binding partner is specific for an activationstate of an activatable element.

The activation state of an individual activatable element is either inthe on or off state. As an illustrative example, and without intendingto be limited to any theory, an individual phosphorylatable site on aprotein can activate or deactivate the protein. The terms “on” and“off,” when applied to an activatable element that is a part of abiological target, are used here to describe the state of theactivatable element, and not necessarily the overall state of thebiological target of which it is a part. Typically, a cell possesses aplurality of a particular protein or other constituent with anactivatable element and this plurality of proteins or constituentsusually has some proteins or constituents whose individual activatableelement is in the on state and other proteins or constituents whoseindividual activatable element is in the off state. Since the activationstate of each activatable element is measured through the use of alabeled protein that recognizes a specific activation state, only thoseactivatable elements in the specific activation state recognized by thebinding element, representing some fraction of the total number ofactivatable elements, will be bound by the labeled protein to generate ameasurable signal. The measurable signal corresponding to the summationof individual activatable elements of a particular type that areactivated in a sample is the “activation level” for that activatableelement in that sample. In some embodiments, activation level ismeasured at the level of single cells, such as a plurality of cells in asample.

Activation levels for a particular activatable element may vary amongindividual cells so that when a plurality of cells is analyzed, theactivation levels follow a distribution. The distribution may be anormal distribution, also known as a Gaussian distribution, or it may beof another type. Different populations of cells may have differentdistributions of activation levels that can then serve to distinguishbetween the populations. In some embodiments, the basis for classifyingcells is that the distribution of activation levels for one or morespecific activatable elements will differ among different phenotypes. Acertain activation level, or more typically a range of activation levelsfor one or more activatable elements seen in a cell or a population ofcells, is indicative that that cell or population of cells belongs to adistinctive phenotype. Other measurements, such as cellular levels(e.g., expression levels) of biomolecules that may not containactivatable elements, may also be used to classify cells in addition toactivation levels of activatable elements; it will be appreciated thatthese levels also will follow a distribution, similar to activatableelements. Thus, the activation level or levels of one or moreactivatable elements, optionally in conjunction with levels of one ormore levels of biomolecules that may not contain activatable elements,of a cell or a population of cells may be used to classify a cell or apopulation of cells into a class. Once the activation level ofintracellular activatable elements of individual single cells is knownthey can be placed into one or more classes, e.g., a class thatcorresponds to a phenotype. A class encompasses a class of cells whereinevery cell has the same or substantially the same known activationlevel, or range of activation levels, of one or more intracellularactivatable elements, or other binding partners detected by the labeledproteins of the invention. For example, if the activation levels of fiveintracellular activatable elements are analyzed, predefined classes thatencompass one or more of the intracellular activatable elements can beconstructed based on the activation level, or ranges of the activationlevels, of each of these five elements. It is understood that activationlevels can exist as a distribution and that an activation level of aparticular element used to classify a cell may be a particular point onthe distribution but more typically may be a portion of thedistribution.

In addition to activation levels of intracellular activatable elements,expression levels of intracellular or extracellular binding partners canbe used alone or in combination with activation states of activatableelements and/or expression levels to classify cells. In someembodiments, other characteristics that affect the status of abiological target may also be used to classify a cell. Examples includethe translocation of binding partners or changes in their turnover ratesand the formation and disassociation of complexes of comprising abinding partner. Such complexes can include multi-protein complexes,multi-lipid complexes, homo- or hetero-dimers or oligomers, andcombinations thereof. Other characteristics include proteolyticcleavage, e.g. from exposure of a cell to an extracellular protease orfrom the intracellular proteolytic cleavage of a binding partner.

In some embodiments, the physiological status of one or more cells isdetermined by examining and profiling the activation level of one ormore activatable elements in a cellular pathway. In some embodiments, acell is classified according to the activation level of a plurality ofactivatable elements. In some embodiments, the activation level of oneor more activatable elements in a sample is correlated with a condition.In some embodiments, the activation level of one or more activatableelements in single cells within the sample is determined Cellularconstituents that may include activatable elements include withoutlimitation, proteins, carbohydrates, lipids, nucleic acids andmetabolites. Upon activation, a change occurs to the activatableelement, such as covalent modification of the activatable element (e.g.,binding of a molecule or group to the activatable element, including butnot limited to, phosphorylation, acetylation, methylation,ubiquitination) or a conformational change. Such changes generallycontribute to changes in particular biological, biochemical, or physicalproperties of the biological target that contains the activatableelement. The state of the biological target that contains theactivatable element is often determined to some degree, though notnecessarily completely, by the state of activation of a particularactivatable element of the biological target. For example, a complex maycomprise a protein having multiple activation sites, and the particularactivation states of these sites may overall determine the activationstate of the protein and/or complex. Additional factors, such as thebinding of other proteins, pH, ion concentration, interaction with othercellular constituents, and the like, can also affect the state of thebiological target. In some embodiments, the activation levels of aplurality of intracellular activatable elements in single cells aredetermined. In some embodiments, at least about 2, 3, 4, 5, 6, 7, 8, 9,10 or more than 10 intracellular activatable elements are determined.

Activation states of activatable elements may result from chemicaladditions or modifications of biomolecules and include biochemicalprocesses such as glycosylation, phosphorylation, acetylation,methylation, biotinylation, glutamylation, glycylation, hydroxylation,isomerization, prenylation, myristoylation, lipoylation,phosphopantetheinylation, sulfation, ISGylation, nitrosylation,palmitoylation, SUMOylation, ubiquitination, neddylation,citrullination, amidation, and disulfide bond formation, disulfide bondreduction. Other possible chemical additions or modifications ofbiomolecules include the formation of protein carbonyls, directmodifications of protein side chains, such as o-tyrosine, chloro-,nitrotyrosine, and dityrosine, and protein adducts derived fromreactions with carbohydrate and lipid derivatives. Other modificationsmay be non-covalent, such as binding of a ligand or binding of anallosteric modulator. In general, the activation level is determined bydetecting binding of a labeled protein that is specific for either themodified activatable element or the unmodified activatable element, suchas a labeled antibody specific to a protein phosphorylated at aparticular site, which antibody does not specifically bind to theunphosphorylated protein.

Examples of proteins that may include activatable elements include, butare not limited to kinases, phosphatases, lipid signaling molecules,adaptor/scaffold proteins, cytokines, cytokine regulators,ubiquitination enzymes, adhesion molecules, cytoskeletal/contractileproteins, heterotrimeric G proteins, small molecular weight GTPases,guanine nucleotide exchange factors, GTPase activating proteins,caspases, proteins involved in apoptosis (e.g. PARP), cell cycleregulators, molecular chaperones, metabolic enzymes, vesicular transportproteins, hydroxylases, isomerases, deacetylases, methylases,demethylases, tumor suppressor genes, proteases, ion channels, moleculartransporters, transcription factors/DNA binding factors, regulators oftranscription, and regulators of translation. Examples of activatableelements, activation states and methods of determining the activationlevel of activatable elements are described in US Publication Number20060073474 entitled “Methods and compositions for detecting theactivation state of multiple proteins in single cells” and U.S. Pat. No.7,393,656 entitled “Methods and compositions for risk stratification”the content of which are incorporate here by reference.

In some embodiments, the binding partner is selected from the groupconsisting of HER receptors, PDGF receptors, Kit receptor, FGFreceptors, Eph receptors, Trk receptors, IGF receptors, Insulinreceptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1,Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70,Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGFβreceptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2,MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Wee1, Casein kinases, PDK1,SGK1, SGK2, SGK3, Akt1, Akt2, Akt3, p90Rsks, p70S6Kinase, Prks, PKCs,PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chk1,Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3, IKKs, Cdks, Jnks, Erks, IKKs,GSK3a, GSK3p, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class 3,mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, Receptor proteintyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptortyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs),Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecularweight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases,Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C,PP1, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins,phosphoinositide kinases, phospholipases, prostaglandin synthases,5-lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffoldproteins, Shc, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP),SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nck, Grb2 associated binder(GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-Cellleukemia family, IL-2, IL-4, IL-8, IL-6, interferon γ, interferon β,suppressors of cytokine signaling (SOCs), Cbl, SCF ubiquitination ligasecomplex, APC/C, adhesion molecules, integrins, Immunoglobulin-likeadhesion molecules, selectins, cadherins, catenins, focal adhesionkinase, p130CAS, fodrin, actin, paxillin, myosin, myosin bindingproteins, tubulin, eg5/KSP, CENPs, β-adrenergic receptors, muscarinicreceptors, adenylyl cyclase receptors, small molecular weight GTPases,H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam,Sos, Dbl, PRK, TSC1,2, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2,Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, Bcl-2,Mcl-1, Bcl-XL, Bcl-w, Bcl-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf,Hrk, Noxa, Puma, IAPs, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7,Cyclin D, Cyclin E, Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP,molecular chaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes,Acetyl-CoAa Carboxylase, ATP citrate lyase, nitric oxide synthase,caveolins, endosomal sorting complex required for transport (ESCRT)proteins, vesicular protein sorting (Vsps), hydroxylases,prolyl-hydroxylases PHD-1, 2 and 3, asparagine hydroxylase FIHtransferases, Pin1 prolyl isomerase, topoisomerases, deacetylases,Histone deacetylases, sirtuins, histone acetylases, CBP/P300 family,MYST family, ATF2, DNA methyl transferases, Histone H3K4 demethylases,H3K27, JHDM2A, UTX, VHL, WT-1, p53, Hdm, PTEN, ubiquitin proteases,urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR)system, cathepsins, metalloproteinases, esterases, hydrolases, separase,potassium channels, sodium channels, multi-drug resistance proteins,P-Gycoprotein, nucleoside transporters, Ets, Elk, SMADs, Rel-A(p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos, Spl, Egr-1, T-bet,β-catenin, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1, FOXO STAT1, STAT 3,STAT 4, STAT 5, STAT 6, p53, WT-1, HMGA, pS6, 4EPB-1, eIF4E-bindingprotein, RNA polymerase, initiation factors, elongation factors.

In some embodiments, the classification of a cell according toactivation level of an activatable element, e.g., in a cellular pathwaycomprises classifying the cell as a cell that is correlated with aclinical outcome. In some embodiments, the clinical outcome is theprognosis and/or diagnosis of a condition. In some embodiments, methodsand compositions are provided for the classification of a cell accordingto the activation level of an activatable element, e.g., in a cellularpathway wherein the classification comprises classifying a cell as acell that is correlated to a patient response to a treatment. In someembodiments, methods and compositions are provided for theclassification of a cell according to the activation level of anactivatable element, e.g., in a cellular pathway wherein theclassification comprises selecting a method of treatment.

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. In someembodiments, the kit comprises one or more elements for carrying out amethod of the invention, the elements in one or more containers. In oneembodiment, a kit for labeling a target protein with a dye or detectablelabel comprises a buffer comprising an amine; one or more reactive dyeor detectable label for labeling one or more target proteins; a storagebuffer; and instruction in one or more languages, for example in morethan one language. The reactive dye or detectable label may be areactive dye, a reactive biotin, a reactive digoxin or a reactiveepitope. The dye can be any of the dyes described herein, including butnot limited to an amine reactive dye. The buffer can be any of thebuffers described herein, including but not limited to a sodiumcarbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Trisbuffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In someembodiments, the buffer is alkaline. In some embodiments, the buffer hasa pH from about 7 to about 10. The amine can be any amine describedherein, including but not limited to Tris. In some embodiments, the dyecomprises one or more activated esters, one or more sulfonates, and/orone or more water-soluble polymers, as described herein. In someembodiments, the buffer and dye are sufficient in amount to permitlabeling of 5 μg to 200 μg of a target protein, such as more than about75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more of thetarget protein in a sample is labeled. The storage buffer can be any ofthe storage buffers described herein, and may comprise a stabilizerand/or a preservative as described herein.

In another aspect, a kit for conjugating a reporter protein to a targetprotein is provided, the kit comprising a first buffer comprising afirst amine as described herein; a second buffer comprising an arylamine; a bifunctional crosslinker comprising an amine-reactive group anda hydrolysis-resistant functional group; one or more reporter proteinsfunctionalized with a pairing hydrolysis-resistant reactive group forlabeling one or more target proteins; a storage buffer; and instructionin one or more languages, for example in more than one language. In someembodiments, the bifunctional crosslinker comprises a succinimidyl esterand an aldehyde group, and the one or more reporter proteins arefunctionalized with aminooxy groups.

In some embodiments, the kit further comprises a separation device forpurifying a target protein before labeling, and/or for purifying alabeled target protein. Methods for purifying protein are known in theart, non-limiting examples of which include protein A-based affinitychromatography, size-exclusion chromatography, and ultra membranefiltration. Commercial kits for protein purification, such as antibodycleaning kits, may also be used for target protein purification.Size-exclusion column chromatography and ultra membrane filtration areuseful for removing stabilizers/preservatives of relatively smallmolecules, such as glycerol, Tris, and amino acids. Ultra membranefiltration may also be used to adjust target protein concentration to adesired value. Ultra membrane filtration can be conveniently and rapidlycarried out on a centrifuge using a commercial ultra membrane filtrationvial. The membranes in such ultra filtration devices typically havedifferent pore sizes, or so-called Molecular Weight Cut-off sizes(MWCO), permitting relatively small molecules to go through whileretaining bigger molecules, such as proteins. In some embodiments, amembrane with a MWCO of about, less than about, or more than about 1, 5,10, 15, 20, 25, 50, 100, or more kD is provided to purify a targetprotein before and/or after labeling. Columns for size exclusionchromatography typically comprise particles or beads having pores of aparticular MWCO. Particles larger than the MWCO pass through the columnfaster than particles at or below the MWCO. In some embodiments, acolumn with a MWCO of about, less than about, or more than about 1, 2,10, 15, 20, 25, 50, 100, or more kD is provided to purify a targetprotein before and/or after labeling.

In some embodiments, the kit further comprises a stain stabilizingreagent for enhancing dye fluorescence, such as by enhancing fluorescentintensity or reducing a rate of decrease in fluorescent intensity. Stainstabilizing reagents are known in the art, non-limiting examples ofwhich include EverBrite (Biotium), Vectashield (Vector Laboratories),and SlowFade Gold (Invitrogen). In some embodiments, fluorescentintensity of a dye in the presence of the stain stabilizing reagent isincreased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, 300%, 400%, 500% or more above the intensity of thedye in the absence of the stain stabilizing reagent. In someembodiments, fluorescent intensity of a dye in the presence of the stainstabilizing reagent is maintained above a threshold level for a timethat is at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, or more minutes. In some embodiments, thethreshold level is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more of thestarting fluorescent intensity.

Tables 1-4 provide examples of dyes useful in the methods, compositions,and kits of the present invention.

TABLE 1 Compound No. Structure* 1

2

3

4

5

6

7

8

9

10 

11 

12 

13 

14 

15 

16 

17 

18 

*Wherein when a dye comprise a carboxylic acid group, it is understoodthat the carboxylic acid group can be readily converted to an activatedester via the use of any of a number of reagents, such as DCC, EDC, andTSTU, so that the carboxylic acid group can react with amine group toform an amide linkage.

TABLE 2 Exemplary compounds of the invention are shown. For simplicity,counter ions are not shown in some cases. Absorption/Emission spectrawere measured in PBS unless otherwise indicated. Absorption/ EmissionCompound Wavelengths No. Structure (nm) 19

548/571 20

559/579 21

529/548 22

603/626 23

622/649 24

622/649 25

638/660 26

27

622/648 28

622/649 29

30

31

32

33

34

35

36

37

38

 599/~620 39

40

592/615 41

627/650 42

642/662 43

660/681 44

683/700 45

680/700 46

593/614 47

578/   48

49

50

51

533/550 52

53

642/662 54

TABLE 3 λ_(abs)/λ_(em) Compound (nm) No. Structure (in pH 7.4 PBS) 55

488/516 56

488/516 57

488/516 58

488/516 59

488/516 60

488/516 61

62

63

529/   64

65

66

490/520 67

490/520 68

490/520

TABLE 4 Com- λ_(abs)/λ_(em) pound (nm) No. Structure (H₂O) 69

457/   70

550/570 71

550/570 72

550/570 73

550/570 74

550/570 75

550/570 76

550/570 77

650/665 78

650/665 79

650/665 80

650/665 81

650/665 82

660/680 83

650/665 84

663/690 85

752/778 86

750/775 87

675/694 88

750/775 89

90

768/788 91

92

635/642 93

94

497/513 95

555/565 96

650/665 97

750/770 98

660/675 99

770/790 100 

680/700 101 

790/810 102 

494/520 103 

104 

501/524 105 

106 

107 

520/546 108 

109 

110 

540/565 111 

112 

113 

114 

115 

116 

488/515 117 

494/520 118 

353/442 119 

346/442 120 

416/465 121 

430/545 122 

350/440 123 

124 

125 

400/424

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1 Analysis of Cells Stained with Secondary Antibody Labeled withCompound No. 126

This example describes the labeling of an antibody with a greenfluorescent sulfonated xanthene dye, and the use of such antibodies forintracellular staining and flow cytometry analysis. The dye used in thisexample, Compound No. 126, absorbs light at about 491 nm and emits lightat about 525 nm. The structure of Compound No. 126 is as follows:

Solutions of goat anti-mouse (GAM) labeled with Compound No. 126 wereprepared using different dye-to-antibody ratios and different antibodyconcentrations in a reaction buffer comprising 10 mM Tris and 50 mMNaHCO₃ (pH 8.3). Detailed labeling reaction conditions are listed inTable 5 below. Labeling reactions were carried out by combining antibodyand dye in reaction buffer followed by 30 minutes of incubation.Reaction buffer comprised 10 mM Tris and 50 mM NaHCO₃ with pH ˜8.3. Eachlabeled GAM solution was diluted in a storage buffer and then used incell staining Storage buffer (˜20 mL) was prepared by mixing 10 mL 75 mMTris pH7 buffer, 200 mg BSA, 150 uL 5% NaN₃, 10 mL glycerol and 10 mglysine.

TABLE 5 Labeling condition Antibody- Amount Labeled Antibody to-dye ofGAM IgG Amount of concentration Amount ratio storage antibody GAM IgG inreaction of dye (ug/ buffer solution (μg) (mg/mL) (nmoles) nmole) (uL)#1 100 1 11.25 8.9:1 300 #2 50 2 11.25 4.4:1 300 #3 20 2 5.6 3.6:1 150#4 20 1 2.8 7.2:1 60 #5 5 1 2.8 1.8:1 60 #6 100 1 9.4 10.6:1  300 #7 502 9.4 5.3:1 300 #8 100 1 7.5 13.3:1  300 #9 50 2 7.5 6.7:1 30

Purified dye-GAM IgG conjugate was also prepared for cell staining as acontrol. The purified antibody conjugate was prepared using a standardantibody conjugation method. Briefly, Compound No. 126 (180 nmoles) in50 uL dimethylformamide (DMF) was added to 1 mg of goat anti-mouse at 1mg/mL in 0.1 mM NaHCO₃ (pH 8.3), followed by incubation at roomtemperature for 1 hour. The reaction solution was subject to G-25Sephadex column equilibrated in and eluted with 1×PBS to result in thepurified antibody conjugate. This is referred to as “purified Dye No.1-GAM” in FIG. 1.

One million Jurkat cells per sample were stained with 0.25 μg mouseanti-human CD3 (BD Biosciences) followed by 1 μg of a dye-GAM IgGconjugate prepared according to the above table or 1 μg of the purifiedGAM conjugate. About 10,000 cells from each sample were analyzed on a BDFACS Calibur flow cytometer (BD Biosciences), and fluorescence wasdetected in the FL1 channel using 488 nm excitation. Noise representsthe average intensity from cells stained with only goat anti-mousesecondary conjugates. Signal represents average fluorescence intensityfrom cells stained with CD3 and labeled goat anti-mouse IgG. The dataare plotted in FIG. 1. The results show that cells stained withantibodies labeled according to the invention show high relativefluorescence (white bars) with low background fluorescence (black bars),and perform comparably to the purified antibody. FIG. 1 alsodemonstrates a lack of correlation between relative fluorescence and thestaining conditions tested, indicating that cell staining is independentof the conditions under which the labeled antibodies are prepared (i.e.,the antibody-to-dye ratio and antibody concentration).

Example 2 Analysis of Cells Stained with Secondary Antibody Labeled withCompound No. 42

This example describes labeling of an antibody with a far-redfluorescent sulfonated xanthene dye (Compound No. 42), and the use ofsuch antibodies for intracellular staining and flow cytometry analysis.

Solutions of goat anti-mouse (GAM) labeled with Compound No. 42 wereprepared using different dye-to-antibody ratios and different antibodyconcentrations in a reaction buffer comprising 10 mM Tris and 50 mMNaHCO₃ (pH 8.3). Detailed labeling reaction conditions are listed inTable 6 below. Labeling reactions were carried out by combining antibodyand dye in reaction buffer followed by 30 minutes of incubation.Reaction buffer comprised 10 mM Tris and 50 mM NaHCO₃ with pH ˜8.3. Eachlabeled GAM solution was diluted in a storage buffer and then used incell staining A typical storage buffer (˜20 mL) was prepared by mixing10 mL 75 mM Tris pH7 buffer, 200 mg BSA, 150 uL 5% NaN₃, 10 mL glyceroland 10 mg lysine.

TABLE 6 Labeling condition Antibody- Amount Labeled Antibody to-dye ofGAM IgG Amount of concentration Amount ratio storage antibody GAM IgG inreaction of dye (ug/ buffer solution (μg) (mg/mL) (nmoles) nmole) (uL)#1 100 1 11.25 8.9:1 300 #2 50 2 11.25 4.4:1 300 #3 20 2 5.6 3.6:1 150#4 20 1 2.8 7.2:1 60 #5 5 1 2.8 1.8:1 60 #6 100 1 9.4 10.6:1  300 #7 502 9.4 5.3:1 300 #8 100 1 7.5 13.3:1  300 #9 50 2 7.5 6.7:1 30

Purified Dye-GAM IgG conjugate was also prepared for cell staining as acontrol. The purified antibody conjugate was prepared using standardantibody conjugation method. Briefly, Compound No. 42 (130 nmoles) in 50uL DMF was added to 1 mg of goat anti-mouse at 1 mg/mL in 0.1 mM NaHCO₃,followed by incubation at room temperature for 1 hour. The reactionsolution was subject to G-25 Sephadex column equilibrated in and elutedwith 1×PBS to result in the purified antibody conjugate. This isreferred to as “purified Dye No. 2-GAM conjugate” in FIG. 2.

Cell staining was performed as in Example 1. Stained samples wereanalyzed on BD FACS Calibur using 635 nm excitation and FL4 detectionchannel. Relative fluorescence signals are shown in FIG. 2. The datashow that cell immunostaining according to the invention and thataccording to the conventional method using a purified antibody conjugateproduced comparable results.

Example 3 Analysis of Cells Stained with Secondary Antibody Labeled withOne of Two Compounds

This example describes labeling of an antibody with either a near-IRfluorescent sulfonated xanthene dye (Compound No. 45) or a near-IRsulfonated cyanine dye (Compound No. 127), and the use of suchantibodies for intracellular staining and flow cytometry analysis.Compound No. 127, absorbs light at about 680 nm and emits light at about698 nm. The structure of Compound No. 127 is as follows:

Solutions of goat anti-mouse (GAM) labeled with Compound No. 45 orCompound No. 127 were prepared using different dye-to-antibody ratiosand different antibody concentrations in a reaction buffer comprising 10mM Tris and 50 mM NaHCO₃ (pH 8.3). Detailed labeling reaction conditionsare listed in Table 7 below. Labeling reactions were carried out bycombining antibody and dye in reaction buffer followed by 30 minutes ofincubation. Reaction buffer comprised 10 mM Tris and 50 mM NaHCO₃ withpH ˜8.3. Each labeled GAM solution was diluted in a storage buffer andthen used in cell staining A typical storage buffer (˜20 mL) wasprepared by mixing 10 mL 75 mM Tris pH7 buffer, 200 mg BSA, 150 uL 5%NaN₃, 10 mL glycerol, and 10 mg lysine.

TABLE 7 Labeled Labeling condition GAM Antibody Amount IgG Amountconcentration Amount of Antibody-to- of storage antibody Compound of GAMin reaction dye dye ratio buffer solution No. IgG (μg) (mg/mL) (nmoles)(ug/nmole) (uL) #16 45 50 1 3.8 13.2:1 300 #17 45 100 0.5 3.8 26.3:1 300#18 45 50 1 5.6  8.9:1 300 #19 45 100 0.5 5.6 17.9:1 300 #20 45 50 1 7.5 6.7:1 300 #21 45 100 0.5 7.5 13.3:1 300 #22 45 50 1 9.4  5.3:1 300 #2345 100 0.5 9.4 10.6:1 300 #24 127 50 1 3.8 13.2:1 300 #25 127 100 0.53.8 26.3:1 300 #26 127 50 1 5.6  8.9:1 300 #27 127 100 0.5 5.6 17.9:1300 #28 127 50 1 7.5  6.7:1 300 #29 127 100 0.5 7.5 13.3:1 300 #30 12750 1 9.4  5.3:1 300 #31 127 100 0.5 9.4 10.6:1 300

Purified GAM IgG conjugates to either Compound No. 54 or Compound No.127 were also prepared for cell staining as a control. The purifiedantibody conjugates were prepared using standard antibody conjugationmethod as described in Example 2. Purified conjugates with Compound No.54 are referred to as “purified Dye No. 3-GAM” in FIG. 3. Purifiedconjugates with Compound No. 127 are referred to as “purified Dye No.4-GAM” in FIG. 4.

Cell staining experiments were conducted similarly to Example 1. Stainedsamples were analyzed on BD FACS Calibur using 635 nm excitation and FL4detection channel. Relative fluorescence signals were plotted in FIG. 3.The data show that cell immunostaining according to the invention andthat according to the conventional method using a purified antibodyconjugate produced comparable results.

Example 4 Analysis of Cells Stained with Secondary Antibody Labeled withCompound No. 128

This example describes labeling of an antibody with a far-redfluorescent sulfonated xanthene dye (Compound No. 128), and the use ofsuch antibodies for intracellular staining and flow cytometry analysis.Compound No. 128, absorbs light at about 630 nm and emits light at about650 nm. The structure of Compound No. 128 is as follows:

Solutions of mouse anti-human CD3 antibody (BD Biosciences) labeled withCompound No. 128 were prepared using different dye-to-antibody ratiosand different antibody concentrations in a reaction buffer comprising 10mM Tris and 50 mM NaHCO₃ (pH 8.3). Detailed labeling reaction conditionsare listed in Table 8 below. Labeling reactions were carried out asdescribed in Example 1. Labeled antibody solutions were used directly incell staining.

TABLE 8 Labeling condition Labeled mouse Amount Antibody anti-human ofconcentration Amount Antibody-to- CD3 antibody antibody in reaction ofdye dye ratio solution (μg) (mg/mL) (nmoles) (ug/nmole) #32 100 0.5 5.617.8:1  #33 50 0.5 5.6 8.9:1 #34 20 0.5 2.8 7.2:1 #35 20 0.5 1.4 14.3:1 #36 5 0.5 1.4 3.6:1

One million Jurkat cells per sample were fixed, permeabilized, andstained with 0.25 μg of one of the labeled mouse anti-human CD3 (BDBiosciences) conjugates prepared according to Table 8. For comparison,commercially available mouse anti-human CD3 antibody pre-labeled withspectrally similar Alexa Fluor 647 (BD Biosciences) was also used tostain the cells. About 10,000 cells from each sample were analyzed on aBD FACS Calibur flow cytometer using 635 nm excitation and fluorescencewas detected in the FL4 channel. Signal represents average fluorescenceintensity of the stained cells (FIG. 4). The bars in FIG. 4 representthe average of the fluorescence intensity of duplicate samples. Theresults show that cells stained with antibodies labeled according to theinvention show fluorescence signals comparable to the commerciallyavailable control, and the cell staining intensity is substantiallyindependent of the conditions under which the labeled antibodies areprepared, such as the antibody-to-dye ratio and antibody concentration.

Example 5 Analysis of Cells Stained with Primary Antibody Labeled withCompound No. 42

This example describes labeling of an anti-β-tubulin antibody with afar-red fluorescent sulfonated xanthene dye (Compound No. 42), and theuse of such antibodies for intracellular staining and flow cytometryanalysis. Mouse anti-β-tubulin IgM primary antibody (BD Biosciences) waslabeled with Compound No. 42 according to the conditions listed in Table9 below. Reaction procedure and storage buffer preparation are asdescribed in Example 1.

TABLE 9 Labeling condition Labeled b- Antibody- Amount tubulin Antibodyto-dye of IgM Amount concentration Amount ratio storage antibody of GAMin reaction of dye (ug/ buffer solution IgG (μg) (mg/mL) (nmoles) nmole)(uL) #37 10 0.5 0.56 17.9:1  60 #38 10 0.5 1.3 7.7:1 60 #39 10 0.5 1.47.2:1 60 #40 10 0.5 0.75 13.4:1  60 #41 10 0.5 1.5 6.7:1 60 #42 10 0.51.88 5.3:1 60

One million Jurkat cells per sample were stained with 0.25 μg of one ofthe labeled anti-β-tubulin IgM antibodies prepared according to Table 9.For comparison, commercially available anti-β-tubulin IgM pre-labeledwith spectrally similar Alexa Fluor 647 (BD Biosciences) was also usedto stain the cells. About 10,000 cells from each sample were analyzed ona BD FACS Calibur flow cytometer using 635 nm excitation andfluorescence was detected in the FL4 channel. Signal represents averagefluorescence intensity of the stained cells (FIG. 5). The results showthat cells stained with antibodies labeled according to the inventionshow improved fluorescence signal over the commercially availablecontrol, and the cell staining intensity is substantially independent ofthe conditions under which the labeled antibodies are prepared, such asthe antibody-to-dye ratio and antibody concentration.

Example 6 Microscopy Analysis of Cells Stained with Primary AntibodyLabeled with Cmpd. No. 42

This examples describes the use of antibodies as prepared in Example 5for cell staining of β-tubulin and analysis under a microscope, ascompared to a commercially available antibody. Hela cells were culturedon glass coverslips, fixed with formaldehyde, permeabilized and blockedwith 2% serum/PBS (blocking buffer). Cells were subsequently stainedwith solution #37 of Table 9 or with a purified mouse β-tubulin IgMprimary antibody labeled with Alexa Fluor® 647 purchased from BDBiosciences. Each coverslip was labeled with 1 μg antibody conjugates in200 uL blocking buffer for 1 hr. Cells were extensively washed with1×PBS and mounted onto glass slides. Images were taken on an Olympusmercury arc lamp microscope immediately after staining (t=0) and at 1minute and 3 minutes after staining Example images are shown in FIG. 6.The results show that cells stained with antibody conjugate of thisinvention are comparable to the purified IgM primary antibody labeledwith Alexa Fluor® 647. Cells stained with Compound No. 42 showedcomparable specific staining and intensity compared to cells stainedwith commercially purchased B-tubulin antibody labeled with Alexa Fluor®647. In addition, the labeled antibodies of the invention demonstrate animproved resistance to photobleaching compared to the commerciallyavailable control.

Example 7 Multi-Color Imaging of Cells Labeled with Compound No. 42

Hela cells were cultured on glass coverslips, fixed and stained withCompound No. 42 conjugated with anti-β-tubulin IgM as in Example 5,CF488A-phalloidin, and DAPI. The cells were stained with 1 μg IgMantibody conjugate in 200 uL serum blocking buffer. Stainings withCF488A-phalloidin and DAPI were carried according to the manufacture'sproduct information sheets (Biotium). The images were taken on a ZeissLSM 510 Meta Confocal system. Example images are shown in FIG. 7 ingreyscale. The images show the dye-antibody labeling technology iscompatible with multi-color imaging with other fluorescent probes.

Example 8 Preparation of Aminooxy-Functionalized R-PE

A R-phycoerythrin (R-PE) suspension containing 22.4 mg of R-PE (ProZyme,Hayward, Calif.) was dialyzed against PBS buffer to remove ammoniumsulfate and then concentrated to about 7 mg/mL (˜3.2 mL) viaultramembrane filtration. The resulting R-PE solution was combined with0.32 mL of a pH 8.4 reaction buffer comprising 0.5 M sodium bicarbonateand 0.1 M Tris and 78 uL (0.46 umole) of 4-formylbenzoic acidsuccinimidyl ester in DMF at 6 mM (prepared by mixing equal amount of4-formylbenzoic acid, triethylamine andO-succinimido-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TSTU) andincubing the combined solution for 15 minutes). The combined solutionwas incubated at room temperature for about 1 hour. Thealdehyde-functionalized R-PE was purified with a G-25 column elutingwith PBS. The purified protein was concentrated to about 6 mg/mL Thefunctionalized R-PE (3.4 mL, 85 nmoles), 300 uL (8.4 umoles) of1,3-bis(aminooxy)propane dihydrochloride dissolved in water and 370 uLof a pH 4 phosphate buffer comprising 100 mM aniline were mixed. Thecombined solution was incubated at room temperature for about 1 hour.The resulting solution was run through a G-25 size exclusion columneluting with PBS buffer. The purified aminooxy-functioned R-PE wasconcentrated to about 5 mg/mL.

Example 9 Preparation of Goat Anti-Mouse IgG Labeled with R-PE

Goat anti-mouse (50 μg, 50 uL at 1 mg/mL), 5 uL of pH 8.4 reactionbuffer comprising 0.5 M sodium bicarbonate and 100 mM Tris, and4-formylbenzoic acid succinimidyl ester prepared above in Example 8 (1.7nmoles) in DMF were combined and then incubated for about 30 minutes.About 25 uL of pH 3.7 phosphate buffer comprising 100 mM aniline andabout 150 μg of the aminooxy-R-PE from Example 8 are combined andincubated at room temperature for about 1 hour. The resulting solutionwas used for cell staining without further purification (Example 10).

Example 10 Flow Cytometry Analysis of Intracellular Staining with R-PESecondary Antibody Conjugates Prepared Under Various ConjugationConditions

Approximately one million Jurkat cells per sample were stained with 0.25μg mouse anti-human CD3 (BD Biosciences) followed by 1 μg of R-PE goatanti-mouse IgG conjugate. The fluorescence of 10,000 cells from eachsample was analyzed on a BD FACS Calibur flow cytometer in the FL2channel (488 nm excitation laser, 564-606 nm band-pass emission filter).Signal represents average fluorescence intensity from cells stained withCD3 and goat anti-mouse IgG. Noise represents the average intensity fromcells stained with only goat anti-mouse secondary conjugates, whichreflects the level of non-specific secondary antibody binding. The datawere plotted in FIG. 8. The results show that cells stained withantibodies labeled according to the invention show high specificfluorescence signal and low non-specific antibody binding.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method for preparing a labeled protein comprising: (a) providing i)an amine-reactive dye and ii) a first amine; (b) combining theamine-reactive dye and the first amine, in a buffer, with a samplesolution comprising a target protein, the target protein comprising asecond amine, to form a combined solution; and (c) allowing the combinedsolution to react, thereby producing the labeled protein; wherein theamine-reactive detectable label reacts more strongly with the secondamine than the first amine.
 2. A method for preparing a labeled proteincomprising: (a) providing i) an amine-reactive detectable label and ii)a first amine; (b) combining the amine-reactive detectable label and thefirst amine, in a buffer, with a sample solution comprising a targetprotein, the target protein comprising a second amine, to form acombined solution; and (c) allowing the combined solution to react,thereby producing the labeled protein; wherein the amine-reactivedetectable label reacts more strongly with the second amine than thefirst amine.
 3. The method of claim 2, wherein the amine-reactivedetectable label is not a dye.
 4. The method of claim 3, wherein theamine-reactive detectable label is a biotin, digoxin, hapten, or anepitope.
 5. A method for preparing a labeled protein comprising: (a)providing i) an amine-reactive dye or amine-reactive detectable labeland ii) a first amine; (b) combining the amine-reactive detectable dyeor amine-reactive detectable label and the first amine, in a buffer,with a sample solution comprising a target protein, the target proteincomprising a second amine, to form a combined solution; and (c) allowingthe combined solution to react for less than 3 hours, thereby producinga labeled protein.
 6. The method of claim 1, wherein said amine-reactivedetectable dye or amine-reactive detectable label has a molecular weightof less than about 5000 Da.
 7. The method of claim 1, wherein saidtarget protein is an antibody, for example an antibody fragment, arecombinant antibody, a non-human antibody, a chimeric antibody, ahumanized antibody, or a fully human antibody.
 8. The method of claim 1,wherein said buffer is selected from the group consisting of: a sodiumcarbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Trisbuffer, a MOPS buffer, a HEPES buffer, or a combination thereof.
 9. Themethod of claim 1, wherein said buffer is alkaline.
 10. The method ofclaim 9, wherein the pH of the buffer is in the range from 7 to
 10. 11.The method of claim 1, wherein the pH of said combined solution isgreater than 7.8.
 12. The method of claim 1, wherein said first amine isa primary or secondary amine.
 13. The method of claim 12, wherein saidfirst amine is tris(hydroxymethyl)aminomethane (Tris).
 14. The method ofclaim 13, wherein said tris(hydroxymethyl)aminomethane is present insaid buffer in a concentration of greater than 20 mM.
 15. The method ofclaim 1, wherein said amine-reactive dye or amine-reactive detectablelabel comprises one or more activated esters.
 16. The method of claim15, wherein said one or more activated esters is N-hydroxy succinimidylester, N-hydroxy sulfosuccinimidyl ester, p-sulfo-tetrafluorophenolester, or a combination thereof.
 17. The method of claim 1, wherein saidamine-reactive dye or amine-reactive detectable label comprises one ormore sulfonate groups.
 18. The method of claim 17, wherein said dyecomprises one or more water-soluble polymer groups.
 19. The method ofclaim 18, wherein said water-soluble polymer group is a polyethyleneglycol.
 20. The method of claim 18, wherein said water-soluble polymergroup has a molecular weight of 300-10000 Daltons, or 450-5000 Daltons.21. The method of claim 17, wherein said buffer comprises anamine-reactive dye, which comprises a dye which is selected from thegroup consisting of: a CF dye, an Alexa Fluor dye, a DyLight Dye, a Cydye, an IRDye, a HiLyte dye, a sulfonated and/or pegylated coumarin dye,a sulfonated and/or pegylated xanthene dye, a sulfonated or/pegylatedcyanine dye, and a sulfonated and/or pegylated pyrene dye.
 22. Themethod of claim 17, wherein said amine-reactive dye has a chemicalstructure according to any of Compounds 1-128.
 23. (canceled)
 24. Themethod of claim 1 or 2 wherein the reaction of step (c) is completed in30 minutes or less.
 25. The method of claim 24, wherein more than 95% ofsaid target protein reacts to form labeled protein.
 26. The method ofclaim 1, wherein the sample solution further comprises a stabilizer. 27.The method of claim 26, wherein said stabilizer is bovine serum albumin,gelatin, glycerol, or a combination thereof.
 28. (canceled) 29.(canceled)
 30. (canceled)
 31. (canceled)
 32. The method of claim 1,wherein said sample solution comprises between about 1 μg and 1000 μg,or between about 5 μg and about 200 μg of said target protein.
 33. Themethod of claim 32, wherein said sample solution comprises between about5 μg and 20 μg of said target protein.
 34. The method of claim 32,wherein said sample solution comprises between about 20 μg and 50 μg ofsaid target protein.
 35. The method of claim 32, wherein said samplesolution comprises between about 50 μg and 200 μg of said targetprotein.
 36. The method of claim 1, wherein the ratio of amount ofprotein in microgram (μg) to the amount of reactive dye in nanomoles(nmol) is from 30:1 to 1:1.
 37. The method of claim 1, wherein saidsample solution further comprises one or more non-target proteins. 38.The method of claim 37, wherein the ratio of the combined weight oftarget and non-target protein in microgram (μg) to the amount ofreactive dye in nanomoles (nmol) is from 30:1 to 1:1.
 39. The method ofclaim 37, wherein the ratio of the weight in microgram (μg) of targetprotein to non-target protein is from about 10:1 to about 1:10.
 40. Themethod of claim 37, wherein said target protein is preferentiallylabeled in the presence of said one or more non-target proteins. 41.(canceled)
 42. (canceled)
 43. The method of claim 1, wherein more than90% of said labeled proteins comprise only one molecule of said dye. 44.The method of claim 1, further comprising adding a storage buffer tosaid labeled protein to produce a stored protein solution. 45.(canceled)
 46. (canceled)
 47. (canceled)
 48. (canceled)
 49. (canceled)50. (canceled)
 51. The method of claim 1, wherein said sample solutionis not pretreated before steps (a) or (b), wherein said pretreatmentcomprises purification of said target protein away from one or moreother components of said sample solution.
 52. The method of claim 51,wherein said purification step comprises one or more of: protein A-basedchromatography, size-exclusion chromatography, and ultra membranefiltration.
 53. A method for staining one or more biological targetscomprising: (a) preparing one or more labeled proteins according to themethod of any of the preceding claims, (b) exposing said one or morebiological targets to said one or more labeled proteins, such that saidlabeled protein binds to said one or more biological targets therebystaining said one or more biological targets.
 54. The method of claim53, wherein step (b) is performed without purification of said one ormore labeled proteins from step (a).
 55. The method of claim 53, withthe proviso that a quencher is not added in step (a).
 56. The method ofclaim 53, wherein said staining takes place in vivo.
 57. The method ofclaim 53, wherein said staining takes place in vitro.
 58. The method ofclaim 53, wherein said biological target is a cell surface protein. 59.The method of claim 53, wherein said biological target is anintracellular protein.
 60. The method of claim 53, wherein two or morelabeled proteins are present, and each of said two or more labeledproteins bind to one or more biological targets.
 61. The method of claim60, wherein each of said two or more labeled proteins comprise differentdyes, such that said exposing step (b) renders said different bindingpartners optically distinguishable.
 62. The method of claim 53, furthercomprising analyzing stained biological targets by flow cytometry,western blot, or by microscopy.
 63. (canceled)
 64. The method of claim53, further comprising visualizing fluorescence of the stained one ormore biological targets.
 65. The method of claim 53, further comprisingdiagnosing a condition of a subject based on analysis of said stainedone or more biological targets.
 66. A method for determining the abilityof a test compound to interact with a test target and thereby induce aneffect in one or more biological targets, the method comprising thesteps of: (a) exposing the test target to a test compound; (b) stainingsaid one or more biological targets according to the method of claim 53;and (c) determining the ability of said test compound to interact withsaid test target from the extent of staining of said one or morebiological targets as performed in step (b).
 67. A kit for preparing alabeled protein comprising: (a) a buffer comprising a first amine; (b)one or more amine-reactive dyes for labeling a target protein, saidtarget protein comprising a second amine; (c) a storage buffer; and, (d)instructions in more than one language.
 68. A kit for preparing alabeled protein comprising: (a) an amine-reactive detectable label; anda first amine; (b) instructions directing a user to combine, in abuffer, the amine-reactive detectable label, the first amine, and asample solution comprising a target protein, the target proteincomprising a second amine, to form a combined solution; and wherein theamine-reactive detectable label reacts more strongly with the secondamine than the first amine.
 69. The kit of claim 68, wherein saidamine-reactive label is an amine-reactive dye. 70.-88. (canceled)
 89. Acomposition comprising a labeled protein substantially free of unlabeledprotein, wherein the composition is prepared by a method of claims 1-52without a purification step being performed subsequent to step (c). 90.A method for preparing a protein-protein conjugate comprising: (a)providing i) an amine-reactive bifunctional crosslinker additionallycomprising a hydrolysis-resistant functional group; and optionally ii) afirst amine; (b) combining, in a first buffer, the amine-reactivebifunctional crosslinker and, if present, the first amine, with a samplesolution comprising a target protein, the target protein comprising asecond amine, to form a first combined solution; (c) allowing thecombined solution to react, thereby producing a target proteinfunctionalized with a hydrolysis-resistant functional group; (d)combining the target protein functionalized with thehydrolysis-resistant functional group with a reporter protein comprisinga hydrolysis-resistant reactive group, wherein said hydrolysis-resistantfunctional group reacts with said hydrolysis-resistant reactive group,thereby forming a protein-protein conjugate; wherein the amine-reactivedetectable label reacts more strongly with the second amine than thefirst amine; and wherein step (d) is performed without purification ofthe combined solution from step (c).
 91. The method of claim 90, whereinthe protein-protein conjugate is exposed to a biological target, suchthat said protein-protein conjugate binds to said biological targetthereby staining said biological target.
 92. The method of claim 90,wherein the first buffer is an aqueous buffer.
 93. The method of claim92, wherein the aqueous buffer has a pH of between about 7 and about 10.94. The method of claim 90, wherein said first amine istris(hydroxymethyl)aminomethane (Tris).
 95. The method of claim 92,wherein said buffer is a bicarbonate buffer.
 96. The method of claim 92,wherein the amine-reactive group is an activated ester, for example aN-hydroxy succinimidyl ester, N-hydroxy sulfosuccinimidyl ester, or ap-sulfo-tetrafluorophenol ester.
 97. The method of claim 92, wherein thehydrolysis-resistant functional group is an aldehyde, ketone, azide,alkyne, phosphine, diene, dienophile, protected hydrazine, or protectedaminooxy.
 98. The method of claim 92, wherein the amine-reactive groupis an activated ester and the hydrolysis-resistant functional group isan aldehyde.
 99. The method of claim 92, wherein said sample solutioncomprises between about 1 μg and 1000 μg, or between about 5 μg andabout 200 μg of said target protein.
 100. The method of claim 92,wherein the ratio of amount of protein in microgram (μg) to the amountof reactive dye in nanomoles (nmol) is from 30:1 to 1:1.
 101. The methodof claim 92, wherein the reporter protein is a fluorescent protein, atandem dye or an enzyme.
 102. The method of claim 101, wherein thereporter protein is a phycobiliprotein, for example R-phycoerythrine.103. The method of claim 92, wherein step (d) is performed in an aqueousbuffer.
 104. The method of claim 103, wherein said aqueous buffer has apH from about 4 to about 10.